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.

Comparative genomics identifies potential virulence factors in Clostridium tertium and C. paraputrificum.

Some well-known Clostridiales species such as Clostridium difficile and C. perfringens are agents of high impact diseases worldwide. Nevertheless, other foreseen Clostridiales species have recently emerged such as Clostridium tertium and C. paraputrificum. Three fecal isolates were identified as Clostridium tertium (Gcol.A2 and Gcol.A43) and C. paraputrificum (Gcol.A11) during public health screening for C. difficile infections in Colombia. C. paraputrificum genomes were highly diverse and contained large numbers of accessory genes. Genetic diversity and accessory gene percentage were lower among the C. tertium genomes than in the C. paraputrificum genomes. C. difficile tcdA and tcdB toxins encoding homologous sequences and other potential virulence factors were also identified. EndoA interferase, a toxic component of the type II toxin-antitoxin system, was found among the C. tertium genomes. toxA was the only toxin encoding gene detected in Gcol.A43, the Colombian isolate with an experimentally-determined high cytotoxic effect. Gcol.A2 and Gcol.A43 had higher sporulation efficiencies than Gcol.A11 (84.5%, 83.8% and 57.0%, respectively), as supported by the greater number of proteins associated with sporulation pathways in the C. tertium genomes compared with the C. paraputrificum genomes (33.3 and 28.4 on average, respectively). This work allowed complete genome description of two clostridiales species revealing high levels of intra-taxa diversity, accessory genomes containing virulence-factors encoding genes (especially in C. paraputrificum), with proteins involved in sporulation processes more highly represented in C. tertium. These finding suggest the need to advance in the study of those species with potential importance at public health level.

High frequency of toxigenic Clostridium difficile and Clostridium perfringens coinfection among diarrheic patients at health care facility-onset (HCFO) and community-onset (CO) centers in Bogotá, Colombia.

BACKGROUND: The aim of this study was to evaluate the frequency of toxigenic C. difficile and C. perfringens infections at health care facility-onset (HCFO) and community-onset (CO), in two health care centers (HCC) in Bogotá, Colombia. A total of 220 stool samples from patients presenting diarrhea acquired at HCFO or CO were analyzed by several PCR tests. RESULTS: We found that 65.5% (n = 144) of the population had C. difficile infection, followed by toxigenic C. difficile with 57.3% (n = 126), and finally toxigenic C. perfringens with a frequency of 32.7% (n = 72). CONCLUSIONS: This study is the first molecular detection and characterization of C. difficile and C. perfringens in HCFO and CO in Latin America and demonstrates a relevant frequency of these two species, including coinfection and strikingly diverse toxigenic profiles, especially in the CO.

Unveiling the Multilocus Sequence Typing (MLST) Schemes and Core Genome Phylogenies for Genotyping Chlamydia trachomatis.

Multilocus sequence typing (MLST) has become a useful tool for studying the genetic diversity of important public health pathogens, such as Chlamydia trachomatis (Ct). Four MLST schemes have been proposed for Ct (data available from Chlamydiales MLST databases). However, the lack of a sole standardized scheme represents the greatest limitation regarding typing this species. This study was thus aimed at evaluating the usefulness of the four MLST schemes available for Ct, describing each molecular marker's pattern and its contribution toward a description of intra-specific genetic diversity and population structure. The markers for each scheme, showed a variable power of dicrimination, exhibiting in some cases over estimation in the determination of Sequence Types (STs). However, individual analysis of each locus's typing efficiency and discrimination power led to identifying 8 markers as having a suitable pattern for intra-specific typing. analyzing the 8 candidate markers gave a combination of 3 of these loci as an optimal scheme for identifying a large amount of STs, maximizing discrimination power whilst maintaining suitable typing efficiency. One scheme was compared against core genome phylogenies, finding a higher typing resolution through the last approach. These results confirm once again that although complete genome data, in particular from core genome MLST (cgMLST) allow a high resolution clustering for Ct isolates. There are combinations of molecular markers that could generate equivalent results, with the advantage of representing an easy implementation strategy and lower costs leading to contribute to the monitoring and molecular epidemiology of Ct.

New Insights into Clostridium difficile (CD) Infection in Latin America: Novel Description of Toxigenic Profiles of Diarrhea-Associated to CD in Bogotá, Colombia.

Clostridium difficile (CD) produces antibiotic associated diarrhea and leads to a broad range of diseases. The source of CD infection (CDI) acquisition and toxigenic profile are factors determining the impact of CD. This study aimed at detecting healthcare facility onset- (HCFO) and community-onset (CO) CDI and describing their toxigenic profiles in Bogotá, Colombia. A total of 217 fecal samples from patients suffering diarrhea were simultaneously submitted to two CDI detection strategies: (i) in vitro culture using selective chromogenic medium (SCM; chromID, bioMérieux), followed verification by colony screening (VCS), and (ii) molecular detection targeting constitutive genes, using two conventional PCR tests (conv.PCR) (conv.16S y conv.gdh) and a quantitative test (qPCR.16s). The CD toxigenic profile identified by any molecular test was described using 6 tests independently for describing PaLoc and CdtLoc organization. High overall CDI frequencies were found by both SCM (52.1%) and conv.PCR (45.6% for conv.16S and 42.4% for conv.gdh), compared to reductions of up to half the frequency by VCS (27.2%) or qPCR.16S (22.6%). Infection frequencies were higher for SCM and conv.16S regarding HCFO but greater for CO concerning conv.gdh, such differences being statistically significant. Heterogeneous toxigenic profiles were found, including amplification with lok1/3 primers simultaneously with other PaLoc markers (tcdA, tcdB or tcdC). These findings correspond the first report regarding the differential detection of CDI using in vitro culture and molecular detection tests in Colombia, the circulation of CD having heterogeneous toxigenic profiles and molecular arrays which could affect the impact of CDI epidemiology.