P. T. Delaplain1, J. Wang1, B. Bell1, A. Grishin1, H. Ford1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA
Introduction:
Although necrotizing enterocolitis (NEC) is believed to be associated with bacterial colonization of the neonatal gut, the exact nature of the relationship between NEC and specific groups of bacteria or characteristics of bacterial populations remains largely elusive. Analysis of bacterial populations using high throughput sequencing of 16S RNA brought only limited success, as the same bacterial species were found in sick and healthy individuals. Our previous studies implicated a clinically relevant strain of Cronobacter muytjensii, but not several other strains of this species, as a causative agent of NEC. This lead us to hypothesize that NEC is caused by a plethora of opportunistic pathogens, which may belong to a variety of bacterial species, whereas colonization with non-pathogenic bacteria may be innocuous or even protective. In the framework of this hypothesis, we are characterizing multiple isolates of one of the most common first colonizers, Enterococcus faecalis, and their role in the pathogenesis of experimental NEC.
Methods:
Neonates obtained from timed pregnant rats sourced from Charles River or Harlan were either breast fed or formula fed. E. faecalis bacteria were isolated from the intestines of 4-day-old rats based on their thermotolerance, ability to grow in the presence of azide, characteristic appearance upon Gram staining, and 16S RNA sequencing. The isolates were further characterized for colony morphology, hemolysis, gelatin liquefaction, sugar utilization, antibiotic resistance, and spectra of DNA HindIII restriction fragments. Isolates that possessed different combinations of the corresponding phenotypic traits were considered different strains. The strains identified were examined for their ability to activate the pro-inflammatory transcription factor NF-κ B in IEC-6 enterocytes using Western blotting with anti-Iκ Bα antibodies.
Results:
21 different strains were identified among the 146 independent isolates of E. faecalis. DNA restriction enzyme digestion revealed two unique genomic patterns. Some, but not all strains activated NF-κ B in IEC-6 cells. In addition, small colony size on standard media seemed to correlate with a preference for anaerobic conditions.
Conclusion:
There is a considerable diversity of E. faecalis even in animals coming from the same specific pathogen-free environment. Some bacterial phenotypes, specifically hemolysis, proteolysis, antibiotic resistance, and ability to trigger inflammatory response in enterocytes, may be causatively associated with pathogenicity in NEC. Further characterization of these bacteria may identify both effective first colonizers as well as potentially pathogenic or protective strains.
