Cytotoxin-producing Klebsiella oxytoca in the preterm gut and its association with necrotizing enterocolitis

Abstract

Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disease of premature infants associated with gut bacterial dysbiosis. Using 16S rRNA-based methods, our laboratory identified an unclassified Enterobacteriaceae sequence (NEC_unk_OTU) with high abundance in NEC fecal samples. We aimed to identify this bacterium and determine its potential role in the disease. NCBI database searches for the 16S sequence, selective culture systems, biotyping and polymerase chain reaction were employed to refine classification of NEC_unk_OTU and identify toxinencoding genes from the index NEC case. Bacterial cytotoxin production was confirmed by mass spectrometry and apoptosis assays. Additional fecal samples from 9 NEC and 5 non-NEC controls were analyzed using similar methods and multi-locus sequence typing (MLST) was performed to investigate clonal relationships and define sequence types of the isolates. NEC_unk_OTU was identified as Klebsiella oxytoca, a pathobiont known to cause antibiotic-associated hemorrhagic colitis, but not previously linked to NEC. Including the index case, cytotoxin-producing strains of K. oxytoca were isolated from 6 of 10 subjects with NEC; in these, the K. oxytoca 16S sequence predominated the fecal microbiota. Cytotoxin-producing strains of K. oxytoca also were isolated from 4 of 5 controls; in these, however, the abundance of the corresponding 16S sequence was very low. MLST analysis of the toxin-positive isolates demonstrated no clonal relationships and similar genetic clustering between cases and controls. These results suggest cytotoxin-producing strains of K. oxytoca colonize a substantial proportion of premature infants. Some, perhaps many, cases of NEC may be precipitated by outgrowth of this opportunistic pathogen.

Improved molecular characterization of the Klebsiella oxytoca complex reveals the prevalence of the kleboxymycin biosynthetic gene cluster

“…to preterm NEC (supported by corresponding clinical observations), with bacterial overgrowth in the intestine linked to pathological inflammatory cascades, facilitated by a 'leaky' epithelial barrier and LPS-TL4 activation. Recent work has demonstrated K. oxytoca isolates recovered from infants with NEC can produce kleboxymycin (TM) and TV (47). Taken together with the results from our study, we suggest specific virulence factors -i.e.…”

Tracking Closely Related Enteric Bacteria at High Resolution in Fecal Samples of Premature Infants Using a Novel rRNA Amplicon

“…There were only 10 reads recovered from the week 1 sample for Twin Z, so that sample was excluded from further analysis. For all other samples in Twins Y and Z, Klebsiella oxytoca (orange bars) and Clostridium perfringens (yellow bars) were present at high levels, both of which have been associated with necrotizing enterocolitis in preterm infants ( 11, 12 ). Many of the samples also included high levels of Streptococcus pasteurianus (gray bars), a Group D Streptococcus sp.…”

“…K. grimontii is a species related to but distinct from K. oxytoca and K. michiganensis ( 15 ). The isolates K1, K7, K8, were cultured from fecal samples obtained from non-twin preterm infants with necrotizing enterocolitis (NEC) ( 11 ) and each contained a unique ASV pattern. Klebsiella ASV 13 and 2 were contained in more than one ASV fingerprint, they were present at high levels in isolate K7 and low levels in Twin Z (8 and 9 weeks).…”

“…(15). The isolates K1, K7, K8, were cultured from fecal samples obtained from non-twin preterm infants with necrotizing enterocolitis (NEC) (11) and each contained a unique ASV pattern. Klebsiella ASV 13 and 2 were contained in more than .…”

“…2x PCR mix was added, and PCR was performed as per instructions to amplify and barcode each sample. Samples were pooled, purified via spin column (Qiagen MinElute, cat # 28004).Individual Klebsiella oxytoca isolatesKlebsiella oxytoca fecal isolates were from non-related preterm infants that developed necrotizing enterocolitis(11). To obtain individual isolates, a loopful of fecal material was inoculated into 5 ml of pre-warmed Lysis Broth (Lennox) broth with vitamin supplementation (ATCC MD-VS).…”