Papers by Pier Sandro Cocconcelli
Microbial Pathogenesis, 2008
Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-born... more Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-borne disease in humans. Several surface proteins have been shown to be essential in the adhesion of L. monocytogenes, and in the subsequent invasion of phagocytes. Because the control of the invasion of host cells by Listeria could potentially hinder its spread in the infected host, we have examined the effects of a protease treatment on the ability of L. monocytogenes to form biofilms and to invade tissues. We have chosen serratiopeptidase (SPEP), an extracellular metalloprotease produced by Serratia marcescens that is already widely used as an anti-inflammatory agent, and has been shown to modulate adhesin expression and to induce antibiotic sensitivity in other bacteria. Treatment of L. monocytogenes with sublethal concentrations of SPEP reduced their ability to form biofilms and to invade host cells. Zymograms of the treated cells revealed that Ami4b autolysin, internalinB, and ActA were sharply reduced. These cellsurface proteins are known to function as ligands in the interaction between these bacteria and their host cells, and our data suggest that treatment with this natural enzyme may provide a useful tool in the prevention of the initial adhesion of L. monocytogenes to the human gut.
Microbial Pathogenesis, 2008
Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-born... more Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-borne disease in humans. Several surface proteins have been shown to be essential in the adhesion of L. monocytogenes, and in the subsequent invasion of phagocytes. Because the control of the invasion of host cells by Listeria could potentially hinder its spread in the infected host, we have examined the effects of a protease treatment on the ability of L. monocytogenes to form biofilms and to invade tissues. We have chosen serratiopeptidase (SPEP), an extracellular metalloprotease produced by Serratia marcescens that is already widely used as an anti-inflammatory agent, and has been shown to modulate adhesin expression and to induce antibiotic sensitivity in other bacteria. Treatment of L. monocytogenes with sublethal concentrations of SPEP reduced their ability to form biofilms and to invade host cells. Zymograms of the treated cells revealed that Ami4b autolysin, internalinB, and ActA were sharply reduced. These cellsurface proteins are known to function as ligands in the interaction between these bacteria and their host cells, and our data suggest that treatment with this natural enzyme may provide a useful tool in the prevention of the initial adhesion of L. monocytogenes to the human gut.
Meat Biotechnology, 2008
except for brief excerpts in connection with reviews or scholarly analysis. Use in connection wit... more except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
SUMMARY The emergence and the spread of resistance to antimicrobials in bacteria pose a threat to... more SUMMARY The emergence and the spread of resistance to antimicrobials in bacteria pose a threat to human and animal health and present a major financial cost. In an effort to decrease the development of resistance various actions have been taken at Community level, including the removal of all antibiotics used for growth promotion purposes from animal feed in 2006. With
Trends in Food Science & Technology, 2010
Microbial Pathogenesis, 2008
Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-born... more Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-borne disease in humans. Several surface proteins have been shown to be essential in the adhesion of L. monocytogenes, and in the subsequent invasion of phagocytes. Because the control of the invasion of host cells by Listeria could potentially hinder its spread in the infected host, we have examined the effects of a protease treatment on the ability of L. monocytogenes to form biofilms and to invade tissues. We have chosen serratiopeptidase (SPEP), an extracellular metalloprotease produced by Serratia marcescens that is already widely used as an anti-inflammatory agent, and has been shown to modulate adhesin expression and to induce antibiotic sensitivity in other bacteria. Treatment of L. monocytogenes with sublethal concentrations of SPEP reduced their ability to form biofilms and to invade host cells. Zymograms of the treated cells revealed that Ami4b autolysin, internalinB, and ActA were sharply reduced. These cellsurface proteins are known to function as ligands in the interaction between these bacteria and their host cells, and our data suggest that treatment with this natural enzyme may provide a useful tool in the prevention of the initial adhesion of L. monocytogenes to the human gut.
Applied and Environmental Microbiology, 2006
Denaturing gradient gel electrophoresis allowed us to monitor total bacterial communities and to ... more Denaturing gradient gel electrophoresis allowed us to monitor total bacterial communities and to establish a pattern of succession between species in vacuum-packaged beef stored at 2 and 8°C for 9 weeks and 14 days. Species-specific PCR was used to confirm the presence of Lactobacillus sakei and Lactobacillus curvatus. Multiplex PCRs using 16S rRNA-specific primers allowed differentiation between Leuconostoc species. These methods provided the desired information about microbial diversity by detecting the main microorganisms capable of colonizing this ecological niche.
Fermentation and drying are among the most ancient food preservation techniques used by man. Deve... more Fermentation and drying are among the most ancient food preservation techniques used by man. Developed through the years, these processes prolonged the storage time of meats (and meat products) and brought favorable changes to their organoleptic properties, with respect to the original substrate.
Journal of Microbiological Methods, 2005
Different PCR-DGGE protocols were evaluated to monitor fermentation process and to investigate ba... more Different PCR-DGGE protocols were evaluated to monitor fermentation process and to investigate bacterial communities developed in two artisanal Argentinean fermented sausages. Bacterial universal primers frequently used in PCR-denaturing gradient gel electrophoresis (DGGE) were evaluated. Lactic acid bacteria (LAB) and staphylococci species isolated from Tucumán sausages were used to determine the experimental conditions for PCR amplification and DGGE differentiation. Total microbial DNA extracted directly from both fermented sausages was subjected to DGGE analysis. PCR-DGGE results were different for each set of primers used. Primers Bact-0124f(GC)-Uni-0515r and V1f(GC)-V1r showed to be efficient to differentiate LAB and Staphylococcus cultures while the set V3f(GC)-Uni-0515r allowed to demonstrate the succession of different Lactobacillus and Staphylococcus species during ripening process. An intense band corresponding to Lactobacillus sakei was observed to be present in both samples. Staphylococcus saprophyticus was only observed in Tucumán sausage while a band identified as Brochothrix thermophacta was detected in Có rdoba sausage. PCR-DGGE analysis of different 16S rDNA amplicons was able to discriminate between LAB and Gram-positive, coagulase-negative cocci, resulting an effective tool to establish the microbiota developed in artisanal dry sausages. D
Animal Research, 2004
The aim of this study was to evaluate the effects of peptide fractions purified from a Saccharomy... more The aim of this study was to evaluate the effects of peptide fractions purified from a Saccharomyces cerevisiae product on the growth of the ruminal bacterium Megasphaera elsdenii. Such compounds have been shown to affect the growth and metabolism of M. elsdenii, the ruminal bacteria which uses lactate the most. A hydrophilic fraction purified by means of HPLC has proven to stimulate cell growth, the production of butyrate (+100%) and valerate (+76.1%) as well as the metabolization of lactate (+ 16.9%). This chromatographic fraction was further characterized and seven positively charged peptides were identified. It was observed that two peptides rich in lysine and histidine were the most effective in stimulating growth (+ 18.5%) and lactate utilization (+ 74.1%) in M. elsdenii. These results show that, in addition to previously studied factors such as the supply of nutrients or oxygen removal, the presence of peptidic fractions is involved in the stimulation of bacterial growth by S. cerevisiae.
Fems Microbiology Letters, 1992
Abstract To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal ... more Abstract To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 × 105 transformants per μg of pSC22 plasmid.
International Journal of Food Microbiology, 2007
A new family of putative signaling molecules having a 2(5H)-furanone configuration has been descr... more A new family of putative signaling molecules having a 2(5H)-furanone configuration has been described in this work. They were released during late exponential or stationary phase in different growth media by some gram-positive bacteria, such as Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus sanfranciscensis, Enterococcus faecalis, and a gram-negative species, i.e. Salmonella enterica. A pair of 2(5H)-furanones called furanones A and B occurred in all the conditioned media (CMs) of the species considered. These two molecules showed similar retention times and their spectral data shared the key fragments to include them in the 2(5H)-furanones family. However, some differences were observed in the mass fragmentation profiles. In particular the use of PCA analysis of all the mass fragments enabled the grouping of furanone A profiles of S. enterica, L. helveticus, L. plantarum, L. paraplantarum, L. sanfranciscensis and E. faecalis in one unique cluster with only few exceptions. On the other hand, the mass fragmentation profiles of furanone B of the major part of the species and strains could be grouped together and were differentiated from those of L. helveticus.
Microbiology-sgm, 2008
Staphylococcus epidermidis CNBL 7032 is a heteroresistant strain, with subpopulations resistant t... more Staphylococcus epidermidis CNBL 7032 is a heteroresistant strain, with subpopulations resistant to vancomycin concentrations up to 32 mg l "1 , which was isolated from cured pork meat. The mechanisms of glycopeptide resistance in this strain were investigated in this study. S. epidermidis CNBL 7032 does not harbour enterococcal transmissible vancomycin-resistance genes. Transmission electron microscopy revealed that resistant subpopulations have a thicker cell wall, and that the increase in cell wall thickness is proportional to vancomycin concentration in the growth medium. Scanning electron microscopy showed that S. epidermidis CNBL 7032 forms a biofilm-like structure when grown in the presence of vancomycin. This food isolate harbours the gene atlE, coding for an autolysin with an adhesive function, which is involved in the first phase of biofilm formation. This study has demonstrated an interaction between atlE expression, biofilm formation and glycopeptide antibiotic resistance; transcription analysis demonstrated that the expression of atlE increased proportionally with the vancomycin concentration in the culture. Insertional inactivation of atlE confirmed the role of the AtlE autolysin in biofilm and vancomycin resistance.
Lactobacillus sanfranciscensis CB1, an important sourdough lactic acid bacterium, can withstand l... more Lactobacillus sanfranciscensis CB1, an important sourdough lactic acid bacterium, can withstand low pH after initial exposure to sublethal acidic conditions. The sensitivity to low pH varied according to the type of acid used. Treatment of Lb. sanfranciscensis CB1 with chloramphenicol during acid adaptation almost completely eliminated the protective effect, suggesting that induction of protein synthesis was required for the acid-tolerance response. Two constitutively acid-tolerant mutants, CB1-5R and CB1-7R, were isolated using natural selection techniques after sequential exposure to lactic acid (pH 32). Two-dimensional gel electrophoresis analysis of protein expression by non-adapted, acid-adapted and acid-tolerant mutant cells of Lb. sanfranciscensis showed changes in the levels of 63 proteins. While some of the modifications were common to the acid-adapted and acid-tolerant mutant cells, several differences, especially regarding the induced proteins, were determined. The two mutants showed a very similar level of protein expression. Antibodies were used to identify heat-shock proteins DnaJ, DnaK,
International Journal of Food Microbiology, 2010
The composition of the bacterial consortia of the smear Italian cheeses and their role on quality... more The composition of the bacterial consortia of the smear Italian cheeses and their role on quality and safety is still poorly understood. The objective of this study was to identify and characterize the bacterial communities present on the surface of five traditional Italian cheeses, Casera Valtellina, Scimudin, Formaggio di Fossa, Gorgonzola and Taleggio. DGGE analysis performed using total DNA obtained from cheese surfaces enabled us to identify the dominant bacterial populations. Bands showing different intensity and identified as Staphylococcus, Micrococcus, Psychrobacter, Enterococcus and Brevibacterium species were detected on the surface of cheeses. The cluster analysis showed that Gorgonzola, Taleggio and Formaggio di Fossa cheeses present high similarity in their surface bacterial composition while major differences in the DGGE profiles were observed in Scimudin and Casera. The molecular taxonomical identification among the Gram positive isolates, reveals the presence of the following bacterial genera: Staphylococcus, Micrococcus, Macrococcus, Enterococcus, Lactobacillus, Carnobacterium, Leuconostoc, Brevibacterium, Corynebacterium, Brochothrix, Bacillus. The combination of culture dependent and independent techniques allowed us to obtain information about the bacterial species covering the surface of five different traditional Italian cheeses.
International Journal of Food Microbiology, 2003
This study assessed the frequency of transfer of two mobile genetic elements coding for virulence... more This study assessed the frequency of transfer of two mobile genetic elements coding for virulence determinants and antibiotic resistance factors, into food associated enterococci during fermentation processes. First, the transfer of the pheromone-inducible pCF10 plasmid, carrying tetracycline resistance and aggregation substance (AS) as virulence factor, between clinical and food strains of Enterococcus faecalis, was investigated in models of cheese and fermented sausage. The experiments demonstrated that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from E. faecalis OG1rf cells to food strain E. faecalis BF3098c and that the plasmid subsequently persisted in these environments. Very high frequency of transfer was observed in sausage (10(-3)/recipient) if compared to cheese (10(-6)) and plate mating (10(-4)). Transconjugants were subsequently verified by PCR. The second transmissible element was the plasmid harbouring the vancomycin resistance (VanA phenotype) from E. faecalis A256. The transfer of this antibiotic resistance to a food strain of E. faecalis was studied in vitro and in food models. Although the transfer of vancomycin resistance was achieved in all the environments, the highest conjugation frequencies were observed during the ripening of fermented sausages, reaching 10(-3) transconjugants/recipient cell. PCR confirmed the transfer of the VanA genotype into a food associated Enterococcus strain. This study showed that even in the absence of selective pressure, mobile genetic elements carrying antibiotic resistance and virulence determinants can be transferred at high frequency to food associated enterococci during cheese and sausage fermentation.
International Journal of Food Microbiology, 2005
The dynamics of the microbial community responsible for the artisanal fermentation of dry sausage... more The dynamics of the microbial community responsible for the artisanal fermentation of dry sausage produced in Argentina was investigated by using classical and molecular approaches. The combined use of RAPD analysis with primers M13, XD9, RAPD1 and RAPD2 and 16S rDNA sequencing were applied to the identification and intraspecific differentiation of 100 strains of lactobacilli and Micrococcaceae. DGGE analysis was used to monitor the dynamic changes in population after total microbial DNA was directly extracted from sausages and subjected to PCR using V3f (GC), Bact-0124f-GC and Univ-0515r primers. The sequence analysis of 16S rDNA of the dominant species was also carried out. Lactobacillus sakei and Lactobacillus plantarum were the dominant lactic acid organisms during the fermentation while Staphylococcus saprophyticus represented the dominant species of Micrococcaceae. It was demonstrated that the ripening process of Argentinean artisanal fermented sausage is driven by a limited number of Lactobacillus and Staphylococcus strains selected from environmental microbiota by the ability to best compete under the prevailing conditions of the ecological niche. The identification of dominant communities present in this artisanal fermented sausage can help in the selection of starter cultures consisting in well adapted strains to the particular production technology.
Canadian Journal of Microbiology, 1986
ABSTRACT Slow milk-coagulating variants were isolated from Lactobacillus helveticus HLM 1, a fast... more ABSTRACT Slow milk-coagulating variants were isolated from Lactobacillus helveticus HLM 1, a fast strain which coagulates milk in 16 h at 42 °C. Variants were isolated after subculturing in reconstituted skim milk or complex broth media. Analysis of plasmid content revealed that in slow variants a 3.5-megadalton plasmid was missing.
International Dairy Journal, 2011
The role played by glutamate dehydrogenase (GDH) in Lactobacillus plantarum was investigated by p... more The role played by glutamate dehydrogenase (GDH) in Lactobacillus plantarum was investigated by preparing a GDH-deficient mutant UC1001G. The growth rate and acidification displayed by this mutant were lower than those of the wild-type UC1001. Compared with UC1001G, UC1001 increased the synthesis of proteins that confer a competitive advantage of cell resistance. When both strains were used as adjunct starter for cheese-making, the highest cell survival was found for UC1001. Cheeses made with the adjunct of UC1001 showed the lowest level of free amino acids and the highest content of volatile organic compounds. Both strains produced high level of g-aminobutyric acid in cheese and under simulated gastrointestinal conditions. The findings of this study provide an evidence of the important role of GDH on growth and survival of Lb. plantarum under different environmental conditions that influences some important functional features of the strain.
Research in Microbiology, 2005
The uvrA gene of Lactobacillus helveticus CNBL1156 coding for subunit A of the excinuclease ABC c... more The uvrA gene of Lactobacillus helveticus CNBL1156 coding for subunit A of the excinuclease ABC complex involved in the nucleotide excision repair mechanism was identified. Analysis of the uvrA locus revealed the presence of three open reading frames, merR, sat and uvrA, which coded respectively for a MerR-like regulatory protein, a putative protein with homology to streptothricin acetyl transferase and for a UvrA protein. RNA analysis by northern blotting and RT-PCR showed that sat and uvrA were transcriptionally coupled. UvrA from L. helveticus contained the conserved domains of bacterial excinuclease A, as well as the two ATP binding sites and the zinc binding domains. The transcriptional activity of uvrA indicated that this gene was activated by exposure to UV radiation and oxidative stress. In addition, we observed that the expression of uvrA was inducible by pH; moreover, the role of UvrA in protection against stress was confirmed by acid adaptation experiments. Pretreatment of cells at pH 5 conferred resistance to H 2 O 2 , suggesting a specific adaptive response to pH-induced DNA damage. The results from this study indicate that UvrA contributes to acid and oxidative tolerance in L. helveticus, and suggest that it plays a role in survival at low pH under normal conditions.
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Papers by Pier Sandro Cocconcelli