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

Figures & data

Figure 1. Identification of cytotoxin-producing K. oxytoca from fecal samples of preterm infants. A, Fecal material from the index NEC case was cultured on m-hydroxybenzoate agar; growth was compared to K. pneumoniae and E.coli DH5α. B, Colonies from (A) were cultured on Klebsiella select agar; growth was compared to toxin-negative K. oxytoca (ATCC) and E.coli DH5α. C, Colony PCR for the presence of pehX and npsA/B was performed to further characterize the isolates; toxin-negative K. oxytoca (ATCC) is shown for comparison. Mass spectrometry was performed on bacterial culture supernatants for the detection of tilimycin (D) and tilivalline (E).

Figure 2. Induction of apoptosis by cytotoxin-producing K. oxytoca. A, Caspase-3 activity was measured in T84 enterocytes following 48 hours of exposure to culture supernatants obtained from toxin-positive or toxin-negative K. oxytoca, or media alone (No Tx). Mean percentages are shown from 3 separate experiments. Analysis was by one-way ANOVA with Tukey’s multiple comparisons test. ***P < 0.001 when compared to No Tx or Toxin-negative. Following 72 hours of exposure as above, T84 enterocytes were visualized by light microscopy (B) or stained with PI and evaluated by flow cytometry (C). Percentages of sub-G1 apoptotic cells are shown.

Figure 3. Relative abundance of K. oxytoca OTU in preterm infant fecal samples by 16S rRNA sequencing. A, Mean percentages of rarified reads for the OTU were significantly greater in NEC subjects harbouring toxin-positive strains compared to NEC cases harbouring toxin-negative strains or the non-NEC controls (toxin-positive or toxin-negative). Data represents the composite abundance of K. oxytoca in samples obtained on an approximate weekly basis during the time periods specified in Table 1. Analysis was by one-way ANOVA with Tukey’s multiple comparisons test. ***P < 0.0001, **P < 0.01 when compared to Tox+ NEC. B, The abundance of the K. oxytoca OTU in NEC subjects harbouring toxin-positive strains relative to postnatal day of life, onset of NEC, and administration of systemic antibiotics.

Table 1. Characteristics of study subjects.

Patient ID	Gender	GAge	B.W.	Mode of delivery	Diet	Abx days	Toxin + K. oxytoca	DOL range of samples	DOL NEC	NEC presentation
Case 1	M	29	1350	CS	HM	2	Yes	8–39	36	Bloody stools, pneumatosis, stage 2 NEC
Case 2	M	24	616	CS	HM	3	Yes	8–81	45	Bloody stools, pneumatosis, stage 2 NEC
Case 3	M	26	1006	CS	HM, FM^#	4	Yes	11–62	73	Bloody stools, pneumatosis, stage 2 NEC
Case 4	M	24	800	VD	HM	7	Yes	6–44	10	Bilious emesis, pneumatosis, pneumoperitoneum, K. oxytoca +  blood culture. 12 cm of ileum resected, stage 3 NEC
Case 5	M	23	700	CS	HM	23	Yes	7–96	32	Early SIP (DOL 8) following ibuprofen for PDA: 6 cm of ileum resected. Later developed stage 2 NEC with abdominal distention and pneumatosis
Case 6	F	23	520	CS	HM, FM^##	35	Yes	15–57	61	Early SIP (DOL 6) with E. coli sepsis: peritoneal drain placed. Later developed abdominal distention, pneumatosis, portal venous gas, stage 3 NEC, died
Case 7	M	25	641	VD	HM	19	No	5–61	50	Abdominal distention, pneumatosis, pneumoperitoneum, stage 3 NEC
Case 8	M	25	680	CS	HM	2	No	9–63	24	Abdominal distention, pneumatosis, stage 2 NEC
Case 9	M	26	516	CS	HM	2	No	13–50	35	Abdominal distention, pneumatosis, acidosis, 14 cm of ileum and left colon resected, stage 3 NEC
Case 10	F	27	1026	CS	HM	2	No	14–58	38	Abdominal distention, pneumatosis, bloody stools, mid-gut volvulus noted at laparotomy and 15 cm ileum resected, stage 3 NEC
Control 1	M	23	570	CS	HM, FM^###	33	Yes	29–49	N/A	N/A
Control 2	M	24	646	VD	HM	35	Yes	13–61	N/A	N/A
Control 3	M	25	485	CS	HM	7	Yes	1–87	N/A	N/A
Control 4	M	26	860	CS	HM	9	No	28–78	N/A	N/A
Control 5	F	27	756	CS	HM	2	Yes	17–79	N/A	N/A

GAge: Gestational age in weeks; B.W.: Birth weight in g; Mode of delivery: Cesarean (CS) or vaginal delivery (VD); Diet: Mother’s own milk, donor human milk (HM) or Formula (FM; ^#Similac Special Care, ^##Enfaport, ^###Elecare); Abx Days: Cumulative days of antibiotic exposure prior to NEC diagnosis or during sample collection in Controls; Toxin + K. oxytoca: Present in subjects fecal samples or not; DOL range of samples/NEC: Day of life interval that samples were obtained on an approximate weekly basis, day of life that the infant developed NEC; SIP: Spontaneous intestinal perforation.

Figure 4. MLST phylogenic tree of toxin-positive K. oxytoca isolates obtained from six NEC cases and four non-NEC controls. Phylogenetic tree is constructed based on seven housekeeping genes of K. oxytoca using neighbor-jointing approach. The numbers at the nodes represent bootstrap confidence values based on 1000 replicates. ST numbers are also indicated. New ST: novel sequence type.