Epidemiological and genetic characterization of Clostridium butyricum cultured from neonatal cases of necrotizing enterocolitis in China

Abstract

Objective: Laboratory-based characterization and traceback of Clostridium butyricum isolates linked to outbreak cases of neonatal necrotizing enterocolitis (NEC) in a hospital in China.

Methods: In total, 37 samples were collected during the NEC outbreak. Classical bacteriological methods were applied to isolate and identify Clostridium spp. Meanwhile, 24 samples collected after an outbreak were similarly tested. All Clostridium isolates were identified to species level as either C. butyricum or C. sporogenes. These isolates were subsequently subtyped using pulsed-field gel electrophoresis (PFGE). Genomic DNA was purified from 2 representative C. butyricum isolates and sequenced to completion.

Results: Of 37 samples collected during the NEC outbreak, 17 (45.95%) were positive for Clostridium spp. One species, C. butyricum, was cultured from 10 samples. Another species cultured from 2 other samples was identified as C. sporogenes. Both of these species were cocultured from 5 samples. Pulsotyping showed that the 15 C. butyricum and the 7 C. sporogenes isolates produced indistinguishable DNA profiles. No NEC cases were reported after disinfection following the outbreak, and all samples collected after the outbreak were negative for Clostridium spp. Whole-genome sequencing (WGS) indicated that sialidase, hemolysin, and enterotoxin virulence factors were located on the chromosomes of 2 C. butyricum isolates.

Conclusions: The outbreak of NEC was epidemiologically linked to C. butyricum contamination within the hospital. This is the first report of an NEC outbreak associated with C. butyricum infection in China.

Fig. 1.

Morphology on Columbia blood agar of C. butyricum.

Fig. 2.

Pulsotypes of 15 C. butyricum and 7 C. sporogenesisolates cultured from samples with SmaI restrictive endonuclease.

Fig. 3.

Major virulence genes of C. butyricum F1-b and F1-5 compared with reference strain C. butyricum KNU-L09 (accession no. NZ_CP013252). Beginning from the inside out represents the following features: GC content (black); GC skew (green and purple); C. butyricum KNU-L09 genome (red); C. butyricum F1-b genome (green); C. butyricum F5-b genome (blue); transport genes (shown in green font), putative mobile element (shown in purple font), phage genes (shown in black font) and virulence factor genes (shown in orange font).

Fig. 4.

Genomic environment analysis of phosphotransferase system (PTS) lactose/transposon element which were extracted from whole-genome sequencing (WGS)-based analysis. EasyFig version 1.0 software was using to show the BLAST results. The IS transposases from left to right: IS1182, IS66, IS200/605, IS91, IS256, IS3 and IS30. PTS sugar transporter subunit including PTS lactose/cellobiose transporter subunit IIA, PTS fructose transporter subunit IIB and PTS sugar transporter subunit IIA.