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

Yinping Dong,Yujie Hu,Di Zhang,Fengqin Li,Ying Li,Séamus Fanning,Séamus Fanning,Scott Nguyen,Yu Cao,Nikunj Maheshwari

doi:10.1017/ice.2019.289

Abstract

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

Highlights

15 samples (40.50%) were contaminated with C. butyricum, as determined by gram staining, microscopic examination, biochemical testing, and 16S rRNA gene sequencing. These positive samples included the leftover powdered infant formula (PIF) sample initially consumed by the neonates linked to the Necrotizing enterocolitis (NEC) cases, feces of NEC patients, swab samples taken from the hands of the attending medical staff, disposable items for neonate daily use, breast milk bags, medical equipment used in the ward (eg, infusion pump, an electrocardiogram (ECG) monitor, stethoscope, telephone, refrigerator, as well as handle and inner wall surfaces of the neonate incubator) (Table 2)
Of 37 samples, 5 (13.5%) including the feces of 2 NEC patients, swab samples taken from the inner wall of a refrigerator, the door handle of the incubator as well as disposable items for daily use were cocontaminated with both C. butyricum and C. sporogenes
This outbreak of NEC occurred in a neonatal ward with evidence of association between the presence of toxic C. butyricum strains recovered in the stool samples taken from neonates and the occurrence of NEC

Summary

Ethics statement

Experiments related to infant fecal specimens were performed in accordance with the written informed consent from each patient’s. 61 samples, including 37 taken from sources linked to the NEC outbreak and 24 others (including 1 breast milk and 23 environmental samples) after disinfectant interventions following the outbreak, respectively, were collected for microbiological analysis. Among these 15 NEC patients, 11 had been discharged originally from the hospital at the time of sample collection. Two C. butyricum isolates cultured from samples of the patient’s stool and a swab taken from the inner wall of NEC-infant’s incubator coded F1-b and F5-b, respectively, were recovered, followed by genomic DNA purification. Nucleotide sequences and statistics regarding 2 C. butyricum isolates were submitted to GenBank

Results

F27 Staff 7

Discussion