High-resolution microbiome analysis reveals exclusionary Klebsiella species competition in preterm infants at risk for necrotizing enterocolitis

Intestinal colonization with Klebsiella has been linked to necrotizing enterocolitis (NEC), but methods of analysis usually failed to discriminate Klebsiella species or strains. A novel ~ 2500-base amplicon (StrainID) that spans the 16S and 23S rRNA genes was used to generate amplicon sequence variant (ASV) fingerprints for Klebsiella oxytoca and Klebsiella pneumoniae species complexes (KoSC and KpSC, respectively) and co-occurring fecal bacterial strains from 10 preterm infants with NEC and 20 matched controls. Complementary approaches were used to identify cytotoxin-producing isolates of KoSC. Klebsiella species colonized most preterm infants, were more prevalent in NEC subjects versus controls, and replaced Escherichia in NEC subjects. Single KoSC or KpSC ASV fingerprinted strains dominated the gut microbiota, suggesting exclusionary Klebsiella competition for luminal resources. Enterococcus faecalis was co-dominant with KoSC but present infrequently with KpSC. Cytotoxin-producing KoSC members were identified in most NEC subjects and were less frequent in controls. Few Klebsiella strains were shared between subjects. We conclude that inter-species Klebsiella competition, within an environment of KoSC and E. faecalis cooperation, appears to be an important factor for the development of NEC. Preterm infants seem to acquire Klebsiella primarily through routes other than patient-to-patient transmission.

Klebsiella species colonized most preterm infants, were more prevalent in NEC subjects versus controls, and replaced Escherichia in NEC subjects. StrainID amplicon sequencing returned a total of 3,462,451 reads with a mean of 24,213 ± 1443 reads per sample. Klebsiella was the predominant genus identified for both cases and controls, accounting for a combined 30.6% of total reads from the two groups (Fig. 1A). In individual samples, the mean number of Klebsiella reads was 10,181 ± 1777 and 6100 ± 1007, respectively, in the NEC and control groups ( Fig. 1B; P = 0.03). When normalizing to total reads, Klebsiella accounted for 36.7 ± 5.6% of reads in the NEC group and 27.7 ± 3.7% of reads in the control group ( Fig. 1C; P = 0.055). For individual subjects, Klebsiella was present at a relative abundance of > 2% in 9 of 10 NEC cases, and in 8 of them the relative abundance was > 7%. Alternatively, Klebsiella was present at a relative abundance of > 2% in 12 out of 20 controls, and in 10 of them the relative abundance was > 7% (P = 0.09 and P = 0.11 for comparisons at > 2% and > 7%, respectively). Additionally, the relative abundance of Escherichia was 9.5 ± 2.4% in the control group and 0.04 ± 0.02% in the NEC group ( Fig. 1D; P = 0.005). There were no significant differences for other major taxa including Enterococcus, Enterobacter, Clostridia, Veillonella, Staphylococcus and Streptococcus between the groups (Fig. 1A). samples. All subjects that harbored Klebsiella at a relative abundance of > 2% (9 NEC and 12 controls) were used in the analysis. By plotting the composite relative abundance of reads assigned to Klebsiella species for each subject, we observed that 4 of 9 NEC cases were heavily dominated by KoSC, while the remaining five were heavily dominated by KpSC ( Fig. 2A). A similar pattern of KoSC or KpSC dominance was found in the controls (Fig. 2B), and in subjects located at both NICU sites. KoSC and KpSC were present together in appreciable amounts in only 4 of 21 subjects (Cases 1 and 3, Controls 2 and 5). Among the other major genera identified, the species-level assignments for all subjects were as follows: To further clarify the relationship between KoSC and KpSC in the 4 subjects with appreciable amounts of both, we determined their relative abundances in successive fecal samples over time and in relation to clinical parameters (Fig. 3). Case 1 demonstrated a pattern of KoSC dominance throughout successive weeks despite detection of low levels of KpSC at later time points; no antibiotics were administered during the time points prior to the development of NEC. Enteral feeds were fortified human milk and the diet was supplemented with carbohydrate/fat powder and liquid protein 8 and 4 days, respectively, before the development of NEC. Case 3 displayed the reverse pattern, with KpSC dominance despite detection of low levels of KoSC in later weeks; no antibiotics were administered during the time points prior to the development of NEC. Enteral feeds were fortified human milk and the diet was supplemented with carbohydrate/fat plus amino acid powder 8 days before the development of NEC. Controls 2 and 5, on the other hand, demonstrated dramatic shifts between KoSC and KpSC over successive weeks. Control 2 received treatment with Meropenem and Rifampin for a non-intestinal infection during the interval period but no dietary changes. Control 5 was receiving feeds of fortified human milk and the diet was supplemented with carbohydrate/fat powder 5 days before an increase in KoSC, and with formula 4 days before the disappearance in KpSC; no antibiotics were provided during the time points.   16 . This approach takes advantage of the fact that each individual Klebsiella genome contains eight copies of a 16S-ITS-23S rRNA operon that can vary in sequence and length. The eight copies produce eight distinct amplicons for a single genome, which vary in length and sequence. Generating multiple amplicons per genome presents the opportunity to use the combination as a 'fingerprint' profile to identify a given strain, even if closely related strains share one or more 16S-23S variants. ASV fingerprint patterns representing distinct KoSC or KpSC strains were present in all 9 NEC cases and 12 controls (Fig. 4A,B). The finding that some ASV groupings contained less than eight ASVs indicates that some strains may harbor duplicate copies 16 ; alternatively, ASVs located distant from the origin of replication or that contain a longer ITS region (e.g., more tRNA genes) might drop off before others. Nevertheless, the ASV fingerprinting patterns were distinct and most strains among the NEC cases and controls were unique, indicating that transmission of dominant strains within the subject populations was not occurring. Case 9 and Control 17 shared two distinct patterns, indicating they were likely colonized with the same KoSC strains at different time points (Fig. 4A), while case 10 and Control 19 shared patterns indicating they were likely colonized with the same KpSC strain (Fig. 4B).
Cytotoxin-producing KoSC members were more prevalent in NEC subjects versus controls. Members of KoSC can harbor a biosynthetic gene cluster responsible for generating the enterotoxins tilimycin and tilivalline 12 , which may facilitate mucosal damage in NEC 10 . To determine if cytotoxin-producing KoSC was more prevalent in NEC cases versus controls, and in KoSC-versus KpSC-dominated microbiomes, we analyzed stool samples using PCR and a selective culture system as described in "Methods". Toxin-positive KoSC members were identified and cultured from 7 of 10 NEC cases and 4 of 20 controls (P = 0.007) ( Table 2; Supplementary Tables S1, S2; Figure S1). Phylogroup assignment of the KoSC isolates demonstrated K. michiganesis and K. grimontii to be dispersed across both NICU sites; whereas, K. oxytoca was localized to one NICU ( Table 2). In one control subject (Control 2), KoSC was not detected by PCR or culture, despite KoSC reads being present in the StrainID analysis; this subject received broad-spectrum antibiotics during specimen collection. In

Discussion
A substantial body of evidence points to LPS-induced TLR4 signaling cascades as causal to the profound intestinal inflammatory response observed in infants with NEC 3 . While gut dysbiosis with Gram-negative bacteria is believed to be a key antecedent event 4,17 , there is uncertainty as to whether gut colonization with particular bacterial species predisposes to the development of NEC. In this regard, it is unlikely that all colonizing  www.nature.com/scientificreports/ Gram-negative bacteria share the same pathogenic potential to incite intestinal damage 18 . Members of the genus Klebsiella have been implicated in previous studies [5][6][7] ; however, specific species identification is generally not possible using phenotypic characteristics or short segments of the 16S rRNA gene (e.g., V4) 9 . Here, we utilized a novel long-read amplicon spanning the 16S and 23S rRNA genes to determine the relative abundance of KoSC and KpSC across NICU subjects at risk for developing NEC. We found that Klebsiella was ubiquitous, colonizing the majority of preterm infants; consistent with previous reports [5][6][7]10,14 , Klebsiella was more prevalent in NEC subjects compared to non-NEC controls. We further reveal a pattern of colonization in which the gut microbiota was heavily dominated by either KoSC or KpSC, suggesting competition between the two for luminal resources.  www.nature.com/scientificreports/ The data provide new insights into the microbial community structure in the preterm gut and how members of the genus Klebsiella may contribute to this catastrophic outcome of prematurity. Klebsiella spp. are opportunistic pathogens that are frequently isolated from hospital environments and are a leading cause of nosocomial infections 8,9 . The healthy human microbiome also serves as a potential reservoir for infection; gut colonization with Klebsiella spp. is a well-established precursor for extraintestinal infection in adults 19,20 . While Klebsiella spread among adults is often multi-strain 19,20 , single strain outbreaks also occur 21 . Our analysis of Klebsiella ASV fingerprint patterns identified specific strains in the fecal microbiota of preterm infants and demonstrated that very few KoSC or KpSC strains were shared between subjects. Partial overlap of ASVs between closely related strains and even phylogroups can occur 16 which reflects taxonomic similarities within the 16S-23S region for each species complex. The ASV fingerprinting enables tracking of individual strains, but whole genome sequencing or PCR for genes outside of the 16S-23S region are necessary to further classify KoSC and KpSC members 22,23 . Despite partial overlap of some ASVs, the fingerprint patterns were distinct and recurring for those harboring the same KoSC and KpSC strain. The absence of dominant strains argues against there being a common environmental reservoir (e.g., sink, bedding). We did not sample parents in our cohort, and, given the broad distribution of strains, it is possible that parental transmission or alternate reservoirs were involved.
Production of the cytotoxins tilimycin and tilivalline by KoSC can cause AAHC in older children and adults [11][12][13] and has been linked to NEC in premature infants 10 . Our analysis detected cytotoxin-producing KoSC in the majority of NEC subjects. Surprisingly, several toxin-producing isolates were recovered from NEC subjects whose gut microbiomes were dominated by KpSC. The pattern of either KoSC or KpSC dominance suggests exclusionary Klebsiella species competition, and the context in which competitive stress could increase the expression of virulence factors 24 warrants further investigation. It is possible that intestinal injury represents collateral damage as the result of competitive mechanisms unleashed during Klebsiella inter-species warfare. In the preterm gut, these processes may be further exacerbated by activation of TLR-4 signaling pathways in response to Klebsiella LPS 2,3,25,26 . Thus, the culmination in the development of NEC could involve competitive microbial communities interacting with bacterial signaling receptors (e.g., TLR4) on the premature intestine.
Members of KoSC utilize a broader array of sugars than KpSC which enables them to outcompete KpSC in murine models of gut colonization 27 , while providing the fermentative energy to support cytotoxin production 28 . In Case 3 and Control 5, an increase in KoSC abundance occurred following an increase in dietary carbohydrates. The development of NEC also occurred in Cases 1 and 3 after an increase in dietary carbohydrates. Preterm infants are at risk for carbohydrate malabsorption because they have under-developed intestinal brush border enzymes 29 . The availability of undigested carbohydrates may serve as the impetus for KoSC and KpSC to engage each other in conflict over valuable resources.
Provision of broad-spectrum antibiotics enhances colonization with KpSC 30 . Along these lines, the dramatic shift in KoSC to KpSC observed in Control 2 corresponded with antibiotic treatment for a non-intestinal infection. Many members of KpSC also exploit the type VI secretion system (T6SS) which can facilitate intra-and inter-species killing depending on environmental cues 31 . Deciphering the contextual factors in the gut in association with diversity of genes regulating sugar utilization, cytotoxin production, anti-microbial resistance and T6SS would assist in establishing predictive patterns of KoSC versus KpSC dominance. www.nature.com/scientificreports/ Escherichia was found to be significantly higher in the non-NEC controls, which could indicate that members of this taxa filled a niche that was occupied by Klebsiella in the cases. Enhanced sugar utilization by members of KoSC is known to facilitate colonization resistance against E. coli 32 ; thus, an alternate explanation is that Escherichia was replaced by Klebsiella. Others have observed an increase in E. coli prior to the onset of NEC 33 , and that specific strains of E. coli are associated with more severe disease compared to Klebsiella-associated NEC 34 . Notably, Ward and colleagues found that E. coli and Klebsiella were the two most abundant species in a cohort of preterm infant with NEC; however, the two were infrequently co-habitants 34 . The outcome of competitive interactions among and between E. coli and Klebsiella are likely dependent on the genetic characteristics of individual strains. At the phylum level, microbial dysbiosis preceding NEC is frequently characterized by an increase in Proteobacteria and decrease in Firmicutes 35 . Nevertheless, some reports have demonstrated that early colonization with Firmicutes, such as Staphylococcus and Enterococcus, enhance the risk of developing NEC at later time points 17,36 . We did not observe any associations between Firmicutes and the development of NEC. The abundance of Bifidobacterium spp. also was very low among the subjects, which is consistent with reports of these communities being uncommon in very and extremely premature infants 37 .
Several reports have linked colonization of Enterococcus with KpSC 38,39 . In the current study, we found that E. faecalis was predominantly associated with KoSC, rather than KpSC. Recently, a broad scale genomic analysis differentiated KoSC from KpSC by the presence of genes involved in a type II system to secrete pullulanase, a debranching enzyme that breaks down complex sugars 40 . Enterococci do not express glycosidases that degrade mucosal polysaccharides, and their carbohydrate utilization is limited to less complex sugars 41 . Therefore, it is possible that E. faecalis co-colonizes with KoSC via cooperative carbohydrate metabolism 42 , whereby E. faecalis takes advantage of pullulanase converting complex polysaccharides into small fermentable sugars. Cytotoxin production by KoSC also may be contributory as tilimycin was recently reported to enhance Enterococcus growth and restrict E. coli colonization 43 .
In summary, StrainID amplicon sequencing and ASV fingerprinting provides improved resolution to differentiate KoSC and KpSC in fecal samples and suggests that the two species are in direct competition in the preterm gut. Untangling the contextual factors, genetic diversity of specific strains and competitive mechanisms that can result in intestinal injury are important to understand how these microbes contribute to this devastating disease of prematurity.

Methods
Study population. Subjects were cared for at two affiliated NICUs in Hartford and Farmington, CT. Infants with a gestational age of less than 32 weeks were enrolled from March 2017 to October 2019; those with known congenital malformations of the intestine or not expected to survive beyond the first week were excluded. Study subjects underwent routine care and informed written consent was obtained from a parent on behalf of their infant. Cases were infants whose clinical courses and radiographic findings were consistent with Bell's stage 2 or 3 NEC 44 . Two control subjects were matched to each NEC case. The study was approved by the Institutional Review Board of Connecticut Children's Medical Center and all research was performed in accordance with relevant guidelines/regulations.

Collection of samples and clinical data.
Fecal samples were collected on an approximate weekly basis using sterile disposable spatulas during diaper changes, placed into sterile containers, and immediately frozen at − 80 °C until processing. Samples collected prior to and up to 2-3 weeks after the diagnosis of NEC were included in the analysis. Samples from control infants were time-matched by the closest chronological age corresponding to case samples. Clinical data were obtained from enrolled infants including demographics, gestational age, mode of delivery, day of life (DOL) of NEC, DOL of sample acquisition, exposure to antibiotics, and diet. StrainID amplicon sequencing. Fecal samples were processed and analyzed as previously described 16 .
Briefly, fecal DNA was purified, PCR amplified, and pooled for sequencing using the Complete StrainID Kit (StrainID set A [barcodes 1 to 96]; Intus Biosciences, Farmington, CT) according to the manufacturer's instructions. Amplicon libraries were created using the SMRTbell express template prep kit 2.0 (catalog number 100-938-900; PacBio). The library was sequenced on a Sequel IIe system (Pacific Biosciences) at the University of Delaware, Delaware Biotechnology Institute Sequencing and Genotyping Center, Newark, DE. The selected reads from each sample were primer trimmed and filtered to reads within the length range of 1900-3000 bp. The trimmed and filtered reads were analyzed manually via a histogram to identify peaks of read lengths that are likely to represent unique amplicons. The corresponding read length ranges (i.e., the 2400-to 2405-bp range from each sample) were passed to DADA2 45 and pooled for ASV inference 16 . A sequence table of ASV abundance per sample was produced as part of the DADA2 output, and a heat map was generated in R using the sequence table. SBanalyzer 2.4 (Intus Biosciences) was used to map ccs reads to the Athena database and assign taxonomic identification 16 . Identification of cytotoxin-producing KoSC in fecal samples. DNA from fecal samples and clinical isolates was extracted using the DNeasy Power Soil Kit and DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD), respectively. Fecal samples were screened by PCR for the KoSC-specific gene pehX and genes encoding enzymes in the cytotoxin biosynthetic pathway (npsA and npsB) 46 . For culturing KoSC, a loopful of fecal material was inoculated into 5 ml of Luria-Bertani (Lennox) broth (LB) and grown for 24 h with shaking at 225 rpm at 37 °C. Serial dilutions of fecal cultures were plated on hydroxybenzoic agar as previously described 10 . Individual KoSC colonies were confirmed as toxin-positive or -negative by PCR (pehX, npsA/B) 10  www.nature.com/scientificreports/ San Diego, CA) at UConn Microbial Analysis, Resources, and Services, Storrs, CT. The sequenced genomes were compared across various KoSC phylogroups and an average nucleotide identity (ANI)/OrthoANI value of 98% or greater indicated a matching phylogroup 28,47 . Isolates that were not analyzed by (ANI)/OrthoANI were classified using a PCR-typing strategy to detect the following genes: npsA, bla OXY-1 , bla OXY-2 , bla OXY-4/6 , orfABC, leupAB, and orfA' 22 . Isolates were further characterized by their ability to metabolize d-melezitose 48 . The primer sequences used were as follows: npsA 5′-GCG CTG TTA TGG TTC CCG T-3′ and 5′-CCG GGC ACG CTT GTT ACA  TC-3′; npsB 5′-TGC AGG GTA CGC TAA ATA TTT TAG CT-3′ and 5′-ACC CAC TTA CTT TGC GTA TAA CCA  AT-3′; pehX 5′-GAT ACG GAG TAT GCC TTT ACG GTG -3′ and 5′-TAG CCT TTA TCA AGC GGA TAC TGG -3′; all other primer sequences for the PCR-typing strategy are as published 22 .
Statistics. The analyses were carried out using GraphPad Prism version 9.3.0 (GraphPad Software, San Diego, CA). Mann-Whitney U test was used for quantitative data and Chi-square test for categorical data. A minimum threshold of 400 reads per sample was used to reduce outliers in the microbiome analysis.

Data availability
StrainID amplicon data and the whole-genome assemblies for KoSC isolates are deposited under Bioproject accession number PRJNA908822.