Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) 10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients’ data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia

Matrix-assisted laser desorption ionization--time of flight mass spectrometry: an emerging tool for the rapid identification of mosquito vectors.

BACKGROUND: The identification of mosquito vectors is typically based on morphological characteristics using morphological keys of determination, which requires entomological expertise and training. The use of protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is increasingly being used for the routine identification of bacteria, has recently emerged for arthropod identification. METHODS: To investigate the usefulness of MALDI-TOF-MS as a mosquito identification tool, we tested protein extracts made from mosquito legs to create a database of reference spectra. The database included a total of 129 laboratory-reared and field-caught mosquito specimens consisting of 20 species, including 4 Aedes spp., 9 Anopheles spp., 4 Culex spp., Lutzia tigripes, Orthopodomyia reunionensis and Mansonia uniformis. For the validation study, blind tests were performed with 76 specimens consisting of 1 to 4 individuals per species. A cluster analysis was carried out using the MALDI-Biotyper and some spectra from all mosquito species tested. RESULTS: Biomarker mass sets containing 22 and 43 masses have been detected from 100 specimens of the Anopheles, Aedes and Culex species. By carrying out 3 blind tests, we achieved the identification of mosquito vectors at the species level, including the differentiation of An. gambiae complex, which is possible using MALDI-TOF-MS with 1.8 as the cut-off identification score. A cluster analysis performed with all available mosquito species showed that MALDI-Biotyper can distinguish between specimens at the subspecies level, as demonstrated for An gambiae M and S, but this method cannot yet be considered a reliable tool for the phylogenetic study of mosquito species. CONCLUSIONS: We confirmed that even without any specific expertise, MALDI-TOF-MS profiling of mosquito leg protein extracts can be used for the rapid identification of mosquito vectors. Therefore, MALDI-TOF-MS is an alternative, efficient and inexpensive tool that can accurately identify mosquitoes collected in the field during entomological surveys

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F ST) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study