Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.(AU)

Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry

Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.(AU)

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.(AU)