AMX exposure significantly increased Klebsiella abundance in simulated gut microbiota
To reveal the effects of AMX treatment on the gut bacterial community, AMX was administrated into SHIME model in Fig. 1A. Before the exposure, the system was initially operated in the absence of AMX for three weeks, then 600 mg/day of AMX was injected into the system for another week, followed by the last two weeks of operation after the drug discontinuance. Fecal samples at 14, 21, 24, 28, 35 and 42 days from the ascending, transverse and descending colon vessels were collected for 16S rRNA gene amplicon sequencing. Specifically, these samples represent samples before AMX exposure, after 600 mg/day of AMX treatment, and after termination of AMX (Fig. 1A). The bacteria compositions at the genus and phylum levels at different sampling points were shown in Fig. 1B and Fig. S1, respectively. In terms of the taxonomic assignment at the phylum level, Bacteroidetes, Proteobacteria, and Firmicutes were the three most dominant bacterial phyla in both ascending (accounting for total percent of 91.7%-98.0%) and transverse colon (77.5%-88.8%), while three most abundant phyla in the descending colon were Proteobacteria, Bacteroidetes, and Synergistetes (81.8%-91.1%) (Fig. S1). Regarding genus level, Bacteroides, Klebsiella, Pseudomonas, and Pyramidobacter were predominant in all three colon regions (Fig. 1B). Linear discriminant analysis effect size (LEfSe) results indicated that AMX exposure caused obvious decreases in Bifidobacterium, Phascolarctobacterium and Parabacteroides (Fig. 1C), in accompanying with significant increases in Klebsiella (opportunistic pathogen, LSD = 5.26) and Bacteroides uniformis (probiotics, LSD = 4.75). As Klebsiella genus is an important antibiotic-resistant pathogen in human gut and its unnormal bloom is often associated with multiple diseases, thus we put more attentions on the increased Klebsiella abundance in this study (Fig. 1D). Furthermore, the significant (p < 0.05) enrichment of Klebsiella was observed for both ascending (increased from 1.6% to 32.9%) and transverse colons (increased from 1.5% to 14.0%). Although the abundance of Klebsiella exhibited a partially reduction after the discontinuance of AMX for 2 weeks, the Klebsiella abundance was still obviously higher compared to AMX-free controls (Fig. 1D). For descending colon, a similar higher Klebsiella abundance was also observed although no statistically significant difference between AMX-exposed treatments and AMX-free controls.
Positive correlations between gene numbers of human disease-related pathways and the Klebsiella abundance
The functional composition of bacterial communities was predicted by PICRUST, gene numbers of human disease-related function pathways in bacterial communities in three regions of the colon at different sampling time were obtained in the heatmap (Fig. 2). The results suggested that with the time increased, gene numbers of human disease-related pathways including cancer, drug resistance, infectious diseases, metabolic diseases and neurosurgery diseases exhibited firstly increased then marginally decreased. Regardless of the colon region, AMX exposure significantly enhanced gene numbers of the above pathways, with the greatest enhancement effect in the ascending colon. For example, compared to C-A2 (AMX-free treatment sample) that collected from the ascending colon before AMX administration, gene numbers of the drug resistance pathways were 3.4-4.4 times enriched in the AMX-A2 (AMX exposed treatment sample) that collected after AMX administration for seven days. Similarly, gene numbers of other pathways including bladder cancer, African trypanosomiasis, pertussis and Alzheimer's disease were nearly 4-5.6 times enriched in the AMX-A2 sample than C-A2. Moreover, these human disease-related pathway gene numbers were still maintained at relatively higher levels (about 1.7-2.4 times enriched than AMX-free controls) after the discontinuance of AMX for two weeks.
To explore the potential factors responsible for the changed gene numbers of human disease-related pathways during the incubation period, pearson correlation analyses were performed between gene numbers of pathways and the abundance of Klebsiella. As shown in Fig. 2, the pathway gene numbers of β-lactam resistance, cationic antimicrobial peptide (CAMP) resistance, and vancomycin resistance all had strong positive correlations with the abundances of Klebsiella (Correlation coefficient R was 0.973, 0.944, and 0.974, respectively; p < 0.001). Similarly, the correlations between gene numbers of other human disease-related pathways like cancer, infectious diseases, metabolic diseases, and neurosurgery diseases and the Klebsiella abundance were also positive. Especially the correlation coefficients of pathway gene numbers of bladder cancer, Chagas disease, type II diabetes mellitus, and Huntington's disease with Klebsiella abundance were 0.974, 0.980, 0.969, and 0.968 (p < 0.001), respectively.
Molecular mechanisms for Klebsiella’s contribution to human diseases
In order to further explore the potential molecular mechanisms for the contribution of Klebsiella to human diseases, we isolated Klebsiella strains from AMX-exposed sample AMX-A2 (the most significant effect on human diseases happened in this sample). Using a Klebsiella-specific culture medium, a total of 8 Klebsiella strains were isolated, within which NKU_KlebA1, NKU_KlebA2, NKU_KlebA4, and NKU_KlebA5 possessed all 5 detected ARGs related to drug resistance pathways (bl2b_tem1 and bl2be_shv2 related to β-lactam resistance; arna and rosb related to CAMP resistance; and vang related to vancomycin resistance) (Table S1). Considering NKU_KlebA1 from AMX-exposed treatment has a close genetic distance with the Klebsiella strains from AMX-free control in the 16S rRNA gene-based phylogenetic tree (Fig. 4D), this strain was chosen for further investigation of its whole-genome. The results demonstrated that K. pneumoniae NKU_KlebA1 possesses a ~ 5,250 kp chromosome containing about 5,880 protein-encoding genes with average lengths of 799 bp and average G+C content of 58.6%, as detailed in Table S2 and Fig. 3A. The mobile elements and metabolic systems of this strain were summarized in supplemental Excel S1. Mobile elements were exogenous genome fragments that help bacteria competing for ecological niche and survival in host, resulting in 20 genomic islands, 5 prophages and 4 clustered regularly interspaced palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) systems found in NKU_KlebA1. Meanwhile, metabolic systems of this strain consists of 3 secondary metabolites synthesize gene clusters and 6 carbohydrate-active enzymes, which could help bacteria utilize different kinds of carbohydrate for survive. As shown in Fig. 3B, human disease-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways annotation revealed that K. pneumoniae NKU_KlebA1 possesses drug resistance, cancer, infectious, immune, metabolic, and neurodegenerative diseases pathways, with antimicrobial drug resistance, bacterial infection disease, and cancers as three most dominant annotated pathways. These results provide the genetic level of evidence for Klebsiella’s plausible contribution to human diseases. Since there are multiple drug resistance and other human disease-related pathways in the genome of Klebsiella, the bloom of Klebsiella from AMX exposure would lead to the enrichment of the corresponding pathways. Furthermore, other human disease-related systems including pathogen-host interactions, antibiotic resistance, virulence factors, and secretion systems were summarized in Fig. 3C-F, and details in supplemental Excel S2. The most dominant pathogen-host interaction was reduced virulence (63%), the most dominant drug resistance was efflux pump conferring antibiotic resistance (43%), and the two most dominant virulence factors were iron uptake system (nonspecific virulence factors, 33%) and adherence (offensive virulence factors, 26%). For secretion systems, NKU_KlebA1 carries all genes belonging to the Type II Secretion System, Sec-SRP Secretion System and Tat Secretion System, and some genes belonging to Type VI Secretion System and Type I Secretion System.
AMX exposure significantly increased the virulence of Klebsiella and its molecular mechanisms
To investigate the AMX’s influence on virulence of Klebsiella, 8 Klebsiella strains were also isolated from AMX-free control sample C-A2 (i.e., before AMX exposure) for comparison. Virulence phenotypes including biofilm formation, serum resistance and animal toxicology of all the 16 Klebsiella isolates were evaluated by crystal violet assay, serum bactericidal assay, and G. mellonella infection assay, respectively. The results demonstrated that Klebsiella strains isolated from AMX exposed sample had stronger biofilm formation (p < 0.001, Fig. 4A), serum resistance (p< 0.01, Fig. 4B), and G. mellonella inhibition capacities (p < 0.05, Fig. 4C) than those from AMX-free sample, suggesting AMX exposure dramatically increased the virulence of Klebsiella.
The phylogenetic tree of all isolated Klebsiella species based on 16S rRNA gene identification revealed that the Klebsiella NKU_Kleb8 (isolated from AMX-free sample C-A2) and NKU_KlebA1 (isolated from AMX exposed sample AMX-A2) were within a close genetic distance (Fig. 4D). Further, the whole-genome analysis of strains NKU_KlebA1 and NKU_Kleb8 also implied that these two strains shared only 8 single nucleotide polymorphisms (SNPs) and 471 insertion-deletions (InDels) (as detailed in supplemental Excel S3A). Furthermore, multilocus sequence typing (MLST) confirmed these two strains both belong to ST22, and average nucleotide identity (ANI) analysis demonstrated the nucleotide identity of these two strains were 99.97% (Fig. S2). Therefore, these results collectively suggested that NKU_KlebA1 might be the evolved strain from NKU_Kleb8 exposed to AMX. Using methods described above, we also found NKU_KlebA1 had a substantially higher level of virulence than NKU_Kleb8 in the term of biofilm formation, serum resistance and G. mellonella infection (Fig. 4E-G).
To further confirm that AMX exposure could significantly increase the virulence of Klebsiella, a strain named NKU_Kleb8A7 was evolved from strain NKU_Kleb8 exposure to 600 mg/day AMX for seven days, and results of virulence shown in Fig. 4E-G also manifested a significantly higher virulence of NKU_Kleb8A7 than NKU_Kleb8 (p < 0.01). We subsequently analyzed the virulence factors of the wild strain NKU_Kleb8 and two evolved strains NKU_KlebA1 and NKU_Kleb8A7. Compared with NKU_Kleb8, 8 new virulence factor genes emerged and 39 genes appeared SNP and InDel sites in NKU_KlebA1 (Excel S3B), and 30 new virulence factor genes emerged and 251 genes mutated with SNP and InDel sites in NKU_Kleb8A7 (Excel S3C), as detailed in supplemental Excel S3D and S3E. The detailed genomic mutations of four major classes of virulence factors in NKU_KlebA1 and NKU_Kleb8A7 were shown in Table 1. In detail, capsule virulence factor cpsI appeared InDel sites in NKU_KlebA1, cpsO and wcbB were new emerged virulence factors in NKU_Kleb8A7, and all the others appeared SNP sites in NKU_Kleb8A7. For fimbriae virulence factors, lpfC, pilB, and pilG had genomic mutations in both evolved strains, and most virulence factors appeared InDel sites in NKU_KlebA1 but appeared SNP sites in NKU_Kleb8A7. LPS virulence factor gtrB mutated in both strains, and all the others were new emerged genes or appeared SNP sites in NKU_Kleb8A7. Mutations in fepA, EntD and iutA were also observed in both strains, and all the other siderophores virulence factors were new emerged genes or appeared SNP sites in NKU_Kleb8A7. Owing to their importance in protein function and disease causing, the potential unintended consequence of these mutations needs a special concern.