Isolation and molecular characterization of Clostridium perfringens from healthy Merino lambs in Patagonia region, Argentina

The presence and molecular characterization of Clostridium perfringens in healthy Merino lambs over a six-month period was investigated in this study. Overall, a high prevalence of C. perfringens was detected, even in day-old lambs. Even though the majority of the isolates were characterized as being of type A, types C and D were also isolated. Furthermore, a high genetic diversity was observed by PFGE among the type A isolates.EEA BarilocheFil: Mignaqui, Ana Clara. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marcellino, Romanela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Ronco, T. Technical University of Denmark, National Veterinary Institute; DinamarcaFil: Pappalardo, Juan Sebastián. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Robles, Carlos Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Nonnemann, B. Technical University of Denmark, National Veterinary Institute; DinamarcaFil: Pedersen, K. Technical University of Denmark, National Veterinary Institute; Dinamarc

MALDI-TOF MS: a diagnostic tool for identification of bacterial fish pathogens

Traditional methods to identify bacterial pathogens involved in fish diseases are often slow and/or costly. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS) is an established, fast, accurate and cost effective protein-based technique for identification of bacterial pathogens in human and veterinary laboratories, and has now also been developed for identification of bacterial fish pathogens. Based on analysis of colonies sampled directly from agar plate cultures, the MALDI-TOF technique generates a protein spectrum which is then compared against spectra, called reference spectra, stored in the MALDI-TOF databases. Since not all bacte-rial fish pathogens are covered in commercial databases, database-libraries are usually constructed locally for relevant reference strains/species. In this study, results from two collaborative projects, involving four European veterinary medicine institutes, are presented, focusing on supplementation of available spectra and validation of the MALDI-TOF MS technique for identification of several bacterial fish pathogens. Flavobacterium psychrophilum, F. columnare, Pseudomonas anguilliseptica, Re-nibacterum salmoninarum, Vibrio anguillarum and Yersinia ruckeri are examples of important bacterial fish pathogens that may be successfully identified to the species level by MALDI-TOF MS. On the contrary, identification of Aeromonassalmonicida to species level requires additional phenotypic or genetic analysis to arrive at a correct identification. In conclusion, MALDI-TOF MS has proven a fast, cost effective and accurate identification method for use in fish bacteriology, with a potential for further improvements

MALDI-TOF MS: A diagnostic tool for identification of bacterial fish pathogens

Traditional methods to identify bacterial pathogens involved in fish diseases are often slow and/or costly. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS) is an established, fast, accurate and cost effective protein-based technique for identification of bacterial pathogens in human and veterinary laboratories, and has now also been developed for identification of bacterial fish pathogens. Based on analysis of colonies sampled directly from agar plate cultures, the MALDI-TOF technique generates a protein spectrum which is then compared against spectra, called reference spectra, stored in the MALDI-TOF databases. Since not all bacte-rial fish pathogens are covered in commercial databases, database-libraries are usually constructed locally for relevant reference strains/species. In this study, results from two collaborative projects, involving four European veterinary medicine institutes, are presented, focusing on supplementation of available spectra and validation of the MALDI-TOF MS technique for identification of several bacterial fish pathogens. Flavobacterium psychrophilum, F. columnare, Pseudomonas anguilliseptica, Re-nibacterum salmoninarum, Vibrio anguillarum and Yersinia ruckeri are examples of important bacterial fish pathogens that may be successfully identified to the species level by MALDI-TOF MS. On the contrary, identification of Aeromonassalmonicida to species level requires additional phenotypic or genetic analysis to arrive at a correct identification. In conclusion, MALDI-TOF MS has proven a fast, cost effective and accurate identification method for use in fish bacteriology, with a potential for further improvements

Evaluation of three sample preparation methods for the direct identification of bacteria in positive blood cultures by MALDI-TOF.

BACKGROUND Patient mortality is significantly reduced by rapid identification of bacteria from sterile sites. MALDI-TOF can identify bacteria directly from positive blood cultures and multiple sample preparation methods are available. We evaluated three sample preparation methods and two MALDI-TOF score cut-off values. Positive blood culture bottles with organisms present in Gram stains were prospectively analysed by MALDI-TOF. Three lysis reagents (Saponin, SDS, and SepsiTyper lysis bufer) were applied to each positive culture followed by centrifugation, washing and protein extraction steps. Methods were compared using the McNemar test and 16S rDNA sequencing was used to assess discordant results. RESULTS In 144 monomicrobial cultures, using ≥2.000 as the cut-off value, species level identifications were obtained from 69/144 (48%) samples using Saponin, 86/144 (60%) using SDS, and 91/144 (63%) using SepsiTyper. The difference between SDS and SepsiTyper was not statistically significant (P = 0.228). Differences between Saponin and the other two reagents were significant (P < 0.01). Using ≥1.700 plus top three results matching as the cut-off value, species level identifications were obtained from 100/144 (69%) samples using Saponin, 103/144 (72%) using SDS, and 106/144 (74%) using SepsiTyper and there was no statistical difference between the methods. No true discordances between culture and direct MALDI-TOF identification were observed in monomicrobial cultures. In 32 polymicrobial cultures, MALDI-TOF identified one organism in 34-75% of samples depending on the method. CONCLUSIONS This study demonstrates two inexpensive in-house detergent lysis methods are non-inferior to a commercial kit for analysis of positive blood cultures by direct MALDI-TOF in a clinical diagnostic microbiology laboratory

Rapid Identification and Susceptibility Testing of Candida spp. from Positive Blood Cultures by Combination of Direct MALDI-TOF Mass Spectrometry and Direct Inoculation of Vitek 2

Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST) without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs) is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS) and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥ 1.5 with two identical consecutive propositions), 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤ 1 double dilution step) was 88.6%. Very major errors (VMEs) (false-susceptibility), major errors (false-resistance) and minor errors (false categorization involving intermediate result) amounted to 33.3% (of resistant isolates), 1.9% (of susceptible isolates) and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding