Integrated genomic epidemiology and phenotypic profiling of Clostridium difficile across intra-hospital and community populations in Colombia

Clostridium difficile, the causal agent of antibiotic-associated diarrhea, has a complex epidemiology poorly studied in Latin America. We performed a robust genomic and phenotypic profiling of 53 C. difficile clinical isolates established from diarrheal samples from either intrahospital (IH) or community (CO) populations in central Colombia. In vitro tests were conducted to evaluate the cytopathic effect, the minimum inhibitory concentration of ten antimicrobial agents, the sporulation efficiency and the colony forming ability. Eleven different sequence types (STs) were found, the majority present individually in each sample, however in three samples two different STs were isolated. Interestingly, CO patients were infected with STs associated with hypervirulent strains (ST-1 in Clade-2). Three coexistence events (two STs simultaneously detected in the same sample) were observed always involving ST-8 from Clade-1. A total of 2,502 genes were present in 99% of the isolates with 95% of identity or more, it represents a core genome of 28.6% of the 8,735 total genes identified in the set of genomes. A high cytopathic effect was observed for the isolates positive for the two main toxins but negative for binary toxin (TcdA+/TcdB+/CDT− toxin production type), found only in Clade-1. Molecular markers conferring resistance to fluoroquinolones (cdeA and gyrA) and to sulfonamides (folP) were the most frequent in the analyzed genomes. In addition, 15 other markers were found mostly in Clade-2 isolates. These results highlight the regional differences that C. difficile isolates display, being in this case the CO isolates the ones having a greater number of accessory genes and virulence-associated factors.


Assembly and genome identification
The reads obtained from the sequencing process were assembled de novo to avoid possible biases, considering the highly dynamic nature of the CD genome. We used the improved protocol for Illumina data designed for prokaryotes 6 , which generates multiple assemblies with Velvet v1.2 7 and VelvetOptimiser v2.2.5 (https://packages.debian.org/buster/velvetoptimiser), followed by an assembly improvement step considering the best N50. The best prediction was then assembled using SSPACE 8 and the spaces between contigs were filled using GapFiller 9 .
The assembled genomes were subjected to a delimiting species step using average identity of nucleotides (ANI) 10 . This tool is available as the anib function in Taxxo v1.0, an R package 11,12 . ANI >95.0 was considered to confirm that the genomes belonged to the same species.
The sequence types (STs) circulating in Colombia were determined by multilocus in silico typing using MLSTcheck 13 , which makes comparisons with the PubMLST database (https://pubmlst.org/cdifficile/) 14 that allow the identification of the seven constitutive gene sequences that are part of the standardized scheme for intra-taxa typing of CD 15 and subsequent assignment of the allelic profiles. Supplementary Fig. S1. Schematic representation of the methodology used to characterize the 53 CD Colombian clinical isolates used in this study. a) Whole-genome analysis of the sequences obtained from the isolates. b) Phenotypic characterization of the isolates. In b panel the cytotoxicity assays are remark in pink color, the minimum inhibitory concentration tests in purple color and the sporulation efficiency/viable spores in green color. SBHI, supplemented brain heart infusion (BHI); UFC, unit-forming colony.

Gcol-A92
Gcol-A93 Antibiotic and antiparasitic that causes the breakdown of DNA, the destabilization of the double helix of bacterial DNA and inhibit synthesis of nucleic acids, inhibiting the adequate synthesis of nucleic acids; it is used for the treatment of infections caused by protozoa and anaerobic bacteria; As a mechanism of resistance, possible alterations of metabolic pathways and the formation of biofilms [17][18][19][20][21] Vancomycin ≤0.25-1 0.

-Glycopeptide
This antibiotic, Inhibits the peptidoglycan synthesis, through the inhibition of the enzymes that make up the biosynthetic route of peptidoglycan of the bacterial cell wall; As possible mechanisms of resistance, mutations have been described in the enzymes of the peptidoglycan synthesis pathway, alterations in the antibiotic target proteins (MurG) and the formation of biofilms 17,[20][21][22] Tetracicline ≤0.25->16 Tetraciclines The inhibition of bacterial protein synthesis (limiting its growth) has been described as a mechanism of action. Currently, this antimicrobial agent is not widely used in the treatment of C. difficile infections due to the resistance events generated. As a possible mechanism of resistance, the transference of the tetM, tetW and tet(44) genes, which are responsible for the synthesis of ribosome protection proteins 20,21,[23][24][25] Erythromycin ≤0.25->4 Macrolides These antimicrobials inhibit protein synthesis in the bacteria, occasionally causing cell death; The transfer of non-conjugative mobile elements (Tn5398, Tn6194, Tn6215), which carry copies of the ermB gene that encodes a 23S RNA methylase and induces resistance (this has been described as a mechanism of resistance) 20,21,26 Rifampin ≤0.5->4 0.0039-0.0157 Ansamicinas As an action mechanism, this antimicrobial agent has been described as a transcription inhibitor, by binding to a central segment (residues 500-580) of the β subunit of the DNA-dependent RNA polymerase. It has activity mainly on Grampositive microorganisms; however, it can also act on Gram-negative, besides having antiviral activity. As a mechanism of resistance, has been described that mutations in rpoB (a gene that codes for the β subunit of bacterial RNA polymerase) 17,20,21,27,28 Ampicillin ≤0.5-4 1-4 β-lactam, penicillin This antimicrobial limit the biosynthesis of the bacterial cell wall, through the inhibition of peptidoglycan synthesis; It has bactericidal activity in both Gram positive and Gram negative. As a mechanism of resistance, genes have been identified within the genome of C. difficile that encode β-lactamase-type proteins and penicillin-binding proteins (PBP), which 17,20,21,29,30 would be generating the mechanisms of resistance Penicilin 0.5-8 1-4 β-lactam, penicillin This antibiotic inhibits the biosynthesis of the cell wall of the bacteria. Its activity is mainly on Gram positive bacteria. The mechanisms of resistance are similar to those described for ampicillin 17,20,21,29,30 Fusidic Acid 0.032->256 Cephalosporin Fusidic acid acts by inhibiting bacterial protein synthesis by blocking the elongation factor G (FE-G), preventing it from binding to the ribosomes and GTP (guanosine triphosphate), thus interrupting the energy supply for the synthesis process. As a mechanism of resistance, mutations have been described in fusA, a gene responsible for the coding of FE-G, a ribosomal translocation enzyme 21,[31][32][33] Clindamycin ≤0.25->8 2-8 Lincosamide As a mechanism of action, the inhibition of bacterial protein synthesis has been described. As for erythromycin, the transfer of the ermB gene has been described as a mechanism of resistance 17,20,21 Moxifloxacin 1->4

1-4 Fluoroquinolone
This antimicrobial is a powerful new generation synthetic agent widely used in human infections; Its mechanism of action consists in the inhibition of DNA gyrase in Gram negative and topoisomerase IV in Gram positive. Mutations in the quinolone resistance determinant region of the gyrA and gyrB genes have been decried as resistance mechanisms 17,20,21,24,30,34 Supplementary Data set S1. Assemblies' details of the CD genomes of Colombian isolates.