Risk analysis of Bacillus spp. isolated from cured pork sausages

This study was undertaken to acquire information about the toxigenic potential of Bacillus strains isolated from eight cured pork sausages obtained from traditional or industrial processings. The application of RAPD-PCR protocols made it possible to identify 52 different biotypes among 220 heat-resistant Gram-positive endospore-forming colonies. The sequence analysis of the 5’ region of 16S rDNA revealed that 36 strains belonged to B. subtilis and 16 to B. pumilus species. No strains belonging to B. cereus species were isolated from the cured sausages analysed. The toxigenic potential of these strains was assayed by PCR analysis and physiological tests to identify the most important B. cereus toxins and virulence factors. No specific PCR fragment was obtained from any of the strains; however, some of them were found positive for hemolytic and lecithinase activity.These preliminary results reassure about the microbiological risk related to the presence of pathogenic Bacillus strains in cured pork sausages analysed even though the hemolytic and lecithinase activities found in some strains suggest that more in-depth analyses need to be carried out

Microbial community dynamics during the Scamorza Altamurana cheese natural fermentation.

The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests. Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening. RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus. The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic. Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z dominated all through the fermentation process. Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo. The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products

eoagulase nega tive-sta phylococci and enterococci in fermented meat products: presence of virulence and antibiotic resistance determinants

This study was undertaken to achieve information on the presence of antibiotic resistance determinants in coagulase negative staphylococci (CNS) and enterococci (Ec) that are a significant component of the fermentation process of cured meat products. Vancomycin resistant Ec and CNS were isolated thorough the production process from raw pork meats to final products and, furthermore, many Ec strains were found to be positive in PCR experiments for jsrA and ace, virulence expression factors

Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF-MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.Dr. Kasthuri Venkateswaran, Dr. Irene Ouoba, Dr. Joseph W. Kloepper, Dr. Cecilie From and Dr. Maria Morea are gratefully acknowledged for providing isolates FO-36bT, SAFN-027, SAFN-037, KL-052, 51-3C and 82-2C; Bs31; SE 49 (AP3) and SE 52 (AP7); FEL 55, UNG22 and MIL46, respectively. Raquel Branquinho was supported by a PhD fellowship (Ref. SFRH/BD/61410/2009) and Clara Sousa by a post-doctoral fellowship (Ref. SFRH/BPD/70548/2010), from FCT (Fundacao para a Ciencia e Tecnologia, Portugal). Hugo Osorio acknowledges the funding from QREN-FEDER through the Operational Program ON. 2 - O Novo Norte. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by the Portuguese Foundation for Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competin

Risk analysis of Bacillus spp. isolated from cured pork sausages

This study was undertaken to acquire information about the toxigenic potential of Bacillus strains isolated from eight cured pork sausages obtained from traditional or industrial processings. The application of RAPD-PCR protocols made it possible to identify 52 different biotypes among 220 heat-resistant Gram-positive endospore-forming colonies. The sequence analysis of the 5’ region of 16S rDNA revealed that 36 strains belonged to B. subtilis and 16 to B. pumilus species. No strains belonging to B. cereus species were isolated from the cured sausages analysed. The toxigenic potential of these strains was assayed by PCR analysis and physiological tests to identify the most important B. cereus toxins and virulence factors. No specific PCR fragment was obtained from any of the strains; however, some of them were found positive for hemolytic and lecithinase activity.These preliminary results reassure about the microbiological risk related to the presence of pathogenic Bacillus strains in cured pork sausages analysed even though the hemolytic and lecithinase activities found in some strains suggest that more in-depth analyses need to be carried out.</p

eoagulase nega tive-sta phylococci and enterococci in fermented meat products: presence of virulence and antibiotic resistance determinants

This study was undertaken to achieve information on the presence of antibiotic resistance determinants in coagulase negative staphylococci (CNS) and enterococci (Ec) that are a significant component of the fermentation process of cured meat products. Vancomycin resistant Ec and CNS were isolated thorough the production process from raw pork meats to final products and, furthermore, many Ec strains were found to be positive in PCR experiments for jsrA and ace, virulence expression factors.</p

Microbiological, compositional, biochemical and textural characterization of Caciocavallo Pugliese cheese during ripening

CaciocavalloPugliesecheese during ripening is reported. Fully ripened cheese contained a total of ca. log 8.0 cfu g−1 mesophilic bacteria and ca. log 6.0 cfu g−1 presumptive staphylococci, while the number of thermophilic and mesophilic rod and coccus lactic acid bacteria varied during ripening. A two-step RAPD-PCR protocol was used to differentiate biotypes. The natural whey starter was composed mainly of Lactobacillus delbrueckii, Lb. fermentum, Lb. gasseri, Lb. helveticus and Streptococcus thermophilus strains. After day 1 of ripening, Lb. delbrueckii became dominant and some strains of Enterococcus durans and E. faecalis appeared. Non-starter lactic acid bacteria, such as Lb. parabuchneri and Lb. paracasei subsp. paracasei formed a large part of the lactic microflora at 42 and 60 d of ripening. The level of pH 4.6-soluble nitrogen increased from the outer to the inner of the cheese and also increased in each section as ripening progressed, attaining values of 18–15%. Urea-PAGE electrophoresis showed that degradation of αs1-casein was more rapid than that of β-casein throughout ripening and the rates at which both caseins were degraded greatly increased from the outside to the inside of the cheese. Based on the primary proteolysis products, both chymosin and plasmin appeared to be active. RP-HPLC profiles of the 70% ethanol-soluble, pH 4.6-soluble nitrogen, showed a large number of peaks, indicating a heterogeneous mixture of proteolytic products. There were both age- and section-related changes in the area of the different peptide peaks. Butyric (C4:0), caproic (C6:0), palmitic (C16:0) and oleic (C18:1) acids were the free fatty acids found at the highest concentrations. The level of short chain fatty acids (e.g., butyric and caproic) decreased from the middle and inner to outer sections of the cheese. Peptidase activity in the curd was pronounced, increased during ripening and varied with the cheese section. The greatest increase of the peptidase activity coincided with a change in the lactic microflora and with the prevalence of non-starter lactic acid bacteria. Microbial esterases were supposed to be active together with rennet paste. Little change in the firmness and fractures stress during maturation were found by textural analyses of the raw cheese. The flowability was similar to that of typical low-moisture Mozzarella cheese, while stretchability was lower. The heat-induced changes in phase angle of CaciocavalloPugliesecheese indicated a phase transition from largely elastic rheological characteristics in unheated cheese to a more viscous and fluid character in melted cheese