High-Resolution Differentiation of Enteric Bacteria in Premature Infant Fecal Microbiomes Using a Novel rRNA Amplicon

Twin pair A/B and Y/Z bacterial community profiles. Fecal samples from two pairs of twins were profiled from week 1 to week 8 (twin pair A/B) or week 9 (twin pair Y/Z) after birth. Twins A and B were admitted to a NICU in a different hospital than twins Y and Z. Samples were obtained during the week indicated on the x axis. Results are shown for each sample as a “100% stacked column,” with the y axis box size indicating the percentage of the total sequences. Pink triangles at the top indicate antibiotic treatment prior to sample collection. The following specific taxa are highlighted with the indicated colored borders: Enterobacter cloacae, Klebsiella pneumoniae, E. coli taxa, Bifidobacterium longum, and Clostridium difficile.

Klebsiella amplicon sequence variant fingerprints. (A) Relative-abundance heat map of ∼2,100- to 2,500-base StrainID amplicon sequence variants (ASVs). Samples sharing Klebsiella strains have similar ASV fingerprint patterns. On the x axis, sample IDs are indicated by brackets, and numbers indicate the week since birth when the sample was obtained. Individual samples are indicated by arrows. “Pc” and “Px” indicate fecal sample ASVs from unrelated preterm infants, classified as K. pneumoniae and K. grimontii, respectively. K1, K7, and K8 represent ASVs from Klebsiella isolates from unrelated preterm infants who developed necrotizing enterocolitis. Reads classified by SBanalyzer as Klebsiella were analyzed using DADA2, resulting in the 54 Klebsiella ASVs listed on the y axis. The ASV number is followed by the four-digit ASV length and the taxonomy assigned for that ASV by SBanalyzer. Each coordinate representing an ASV in a sample was colored according to the relative number of reads. Possible species-level taxonomic assignments for the fingerprint in each sample based on the best mapping results on the y axis are indicated by the label colors on the x axis for K. pneumoniae, K. oxytoca/K. michiganensis, and K. grimontii. (B) Klebsiella ASVs in V1 maintain a conserved stem-loop structure. All 54 Klebsiella ASVs were aligned in Jalview (40), with each base numbered according to the alignment and shaded by percent identity. Dark blue variant bases match the most abundant variations, and white indicates a rare variant base. Bases 60 to 110 are indicated at the top of the alignment, part of the 16S V1 region containing a conserved stem-loop based on the E. coli 2-D structure (41). The 5′ stem, loop, and 3′ stem are marked above the alignment by pink, black, and yellow boxes, respectively. Only three variant structures were identified at the V1 loop, shown at the right. Brackets 1 and 2 indicate the ASVs with variants 1 and 2, respectively. Arrow 3 indicates ASV 8, the only ASV with a variant 3 sequence. The pink, black, and yellow regions of the variant structures match the sequence in the alignment window. The red bases in the structures indicate Klebsiella-specific sequences common to these variants that differ from the E. coli structure. The red arrows in variants 1 and 2 indicate a deletion from the E. coli and variant 3 structures. The blue ovals indicate base variations between the aligned Klebsiella ASVs.

Enterobacter amplicon sequence variant fingerprints. (A) Enterobacter ASVs were inferred from longitudinal samples at the indicated weeks from reads classified as Enterobacter by SBanalyzer from twins A and B and an additional individual, infant 961, indicated by brackets. All ASVs were mapped to the NCBI database, and the closest-matching taxonomies were selected and are displayed on the y axis to the right. The relative number of each of the ASVs in each sample is indicated by shading. Points 6 and 6r in twin B are technical repeats of the week 6 fecal sample. (B) Enterobacter ASV mapping to the 2-D 16S rRNA structure. The 12 Enterobacter ASVs inferred from twin A, twin B, and infant 961 using DADA2 were aligned against each other in Jalview (40), with each base numbered according to the alignment and shaded by percent identity. Dark blue variant bases match the most abundant variations, and white indicates a rare variant base. The stem-loop rRNA structures are based on E. coli (41). The selected sequences shown in the middle were mapped to the 16S gene structure from bases 955 to 1015 (the V6 stem-loop [green box above the sequence]) and bases 1180 to 1230 (region between V7 and V8 [light blue box above the sequence]). Identical sequences across all ASVs are shown in black text with dark blue highlighting. Variant sequences are shaded and boxed with different colors, indicating the text color of the variant bases in the corresponding structures. The structure above the alignments represents all twin A/B ASV structures, which are identical in the regions shown. There are three variant structures in this sequence shared within the infant 961 samples, shown below the alignments. The variations in the V7-V8 region from bases 1190 to 1191 form a base-paired structure with the V6 region ∼200 bases away at bases 960 to 961.

E. coli amplicon sequence variant fingerprints. (A) E. coli ASVs were inferred from longitudinal samples at the indicated weeks from reads classified as E. coli by SBanalyzer from twin pair A/B and twin pair Y/Z. Twins A and B were admitted simultaneously to hospital 1, and twins Y and Z were admitted simultaneously to hospital 2, as indicated. The 8 ASVs obtained from the samples using DADA2 were mapped to the NCBI database, and the closest-matching taxonomies were selected and are displayed to the right. ASVs in green text indicate perfect matches to the indicated NCBI region (reg.). “Novel” in bold text indicates an ASV that did not match perfectly to any sequence in the NCBI database but was present in multiple samples. The twin ID and the week that the sample was collected are indicated at the bottom. The relative number of each of the ASVs in each sample is indicated by shading, where dark red indicates more reads and blue indicates fewer. (B) E. coli ASV structure at the V6 stem-loop. E. coli ASVs were inferred from twin pair A/B and twin pair Y/Z using DADA2, and the eight resulting ∼2,300- to 2,400-base 16S-ITS-23S ASVs were aligned against each other in Jalview (40). A portion of the alignment from bases 948 to 1103 containing the V6 region is shown, with identical sequences across all ASVs in black text with dark blue highlighting and ASV-specific variations shaded in lighter colors. The yellow and red boxes above the alignment indicate the V6 stem-loop sequences shaded in the structures pictured immediately below the alignment in the middle. The 2-D structures of the aligned V6 regions of each ASV sequence were mapped to the E. coli 16S gene structure (41) from bases 988 to 1050; bases were numbered according to the alignment. Variant sequences from the alignment are depicted in bold red text. Yellow and red shading in the structures corresponds to the yellow and red boxes above the aligned sequences. The ASVs containing the exact stem-loop V6 sequence are listed below each structure. ASVs with NCBI BLAST searches that produced perfect matches to the entire V6 sequence are listed immediately below the structure in bold green underlined text. The NCBI BLAST strain ID, genomic region, and ASV length for the perfect full-length ASV match containing the V6 sequence are listed immediately below the bold green text. The individual twin samples containing ASVs that include each V6 sequence are listed at the bottom in the outlined boxes.

Uniquely identifiable Klebsiella, Enterobacter, and E. coli genomes in the Athena database. Amplicon sequences were compared for 458 Klebsiella, 109 Enterobacter, and 187 E. coli genome entries in the Athena database. Each Klebsiella and Enterobacter genome ID contained up to eight contiguous 16S-23S gene pairs, and E. coli contained up to seven, from which the sequences of the V4, V1-V3, V1-V9, and StrainID amplicons were extracted. A genome ID was considered unique if it contained either a unique amplicon or a unique combination of amplicons. The percentage of uniquely identifiable genomes is shown on the y axis, and the taxonomy and amplicon type are shown on the y axis.

Whole-genome and 16S phylogenetic analyses. Ten K. oxytoca strains represented in the Athena database were compared with 11,300 strains in the TYGS database to identify closely related strains. The partial trees shown include all branches needed to place the 10 “user strains” selected from the Athena database. Colored boxes are used to indicate groups of genomes that sorted together. (a) Whole-genome phylogeny. The inset box indicates how the input Athena strains were identified by TYGS. The tree with detailed relationships was inferred with FastME 2.1.6.1 (37) from GBDP distances calculated from genome sequences. The numbers above branches are GBDP pseudobootstrap support values of >60% from 100 replications, with an average branch support of 85.5%. (b) 16S phylogeny tree inferred with FastME 2.1.6.1 from GBDP distances calculated from 16S rRNA gene sequences. The numbers above branches are GBDP pseudobootstrap support values of >60% from 100 replications, with an average branch support of 53.3%. GenBank accession numbers are in parentheses. SSU, small subunit.