Prevalence, distribution, and molecular characterization of Salmonella recovered from swine finishing herds and a slaughter facility in Santa Catarina, Brazil.

Projeto: 04.10.06.001

From DNA sequence to application: possibilities and complications

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF’s) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

Viability of a Five-Strain Mixture of Listeria monocytogenes in Vacuum-Sealed Packages of Frankfurters, Commercially Prepared with and without 2.0 or 3.0% Added Potassium Lactate, during Extended Storage at 4 and 10° C†‡

The viability of Listeria monocytogenes was monitored on frankfurters containing added potassium lactate that were obtained directly from a commercial manufacturer. Eight links (ca. 56 g each) were transferred aseptically from the original vacuum-sealed bulk packages into nylon-polyethylene bags. Each bag then received a 4-ml portion of a five-strain mixture of the pathogen. Frankfurters containing 2.0 or 3.0% potassium lactate were evaluated using 20 CFU per package, and frankfurters containing 3.0% potassium lactate were evaluated using 500 CFU per package. The packages were vacuum-sealed and stored at 4 or 10°C for up to 90 or 60 days, respectively. During storage at 4°C, pathogen numbers remained at about 1.6 log10 CFU per package over 90 days in packages containing frankfurters with 2.0% potassium lactate that were inoculated with about 20 CFU. In packages containing frankfurters with 3.0% potassium lactate that were inoculated with about 20 CFU and stored at 4°C, pathogen numbers remained at about 1..

Isolation of Escherichia coli O157:H7 from Intact Colon Fecal Samples of Swine1

Escherichia coli O157:H7 was recovered from colon fecal samples of pigs. Polymerase chain reaction confirmed two genotypes: isolates harboring the eaeA, stx1, and stx2 genes and isolates harboring the eaeA, stx1, and hly933 genes. We demonstrate that swine in the United States can harbor potentially pathogenic E. coli O157:H7

Retail survey of Brazilian milk and minas frescal cheese and a contaminated dairy plant to establish prevalence, relatedness, and sources of listeria monocytogenes isolates.

A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, “brand F” of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil

Fate of Listeria monocytogenes and Shiga Toxin-Producing Escherichia coli on Bresaola Slices During Storage

The viability of multistrain cocktails of genetically marked strains of Listeria monocytogenes and Shiga toxin-producing Escherichia coli (STEC) were separately monitored on slices of one brand of a commercially produced bresaola (ca. pH 6.7 and aw 0.899) during extended storage at refrigeration and abusive temperatures. Two slices (ca. 8 g each; ca.10.2 cm wide, ca. 11 cm long) of bresaola were layered horizontally within a nylon-polyethylene bag. The outer surface of each slice was inoculated (50μL total; ca. 3.5 log colony-forming units [CFU]/package) with a rifampicin-resistant (100μg/mL) cocktail of either L. monocytogenes (5 strains) or STEC (8 strains). Bags were vacuum-sealed and then stored at 4°C or 10°C for 180 or 90 d, respectively. In each of 5 trials, 3 bags were analyzed for pathogen presence at each sampling interval via the US Department of Agriculture–Agricultural Research Service package rinse method. In general, levels of L. monocytogenes and STEC decreased by 3.0 and 2.4 log CFU/package, respectively, after 180 d when bresaola was stored at 4°C. When bresaola was stored at 10°C for 90 d, levels of L. monocytogenes and STEC decreased by 2.4 and 3.1 log CFU/package, respectively. Thus, the sliced bresaola evaluated herein did not provide a favorable environment for either persistence or outgrowth of surface-inoculated cells of L. monocytogenes or STEC

Evaluation of post-fermentation heating times and temperatures for controlling Shiga toxin-producing Escherichia coli cells in a non-dried, pepperoni-type sausage

Coarse ground meat was mixed with non-meat ingredients and starter culture (Pediococcus acidilactici) and then inoculated with an 8-strain cocktail of Shiga toxinproducing Escherichia coli (ca. 7.0 log CFU/g). Batter was fine ground, stuffed into fibrous casings, and fermented at 35.6°C and ca. 85% RH to a final target pH of ca. pH 4.6 or ca. pH 5.0. After fermentation, the pepperoni- like sausage were heated to target internal temperatures of 37.8°, 43.3°, 48.9°, and 54.4°C and held for 0.5 to 12.5 h. Regardless of the heating temperature, the endpoint pH in products fermented to a target pH of pH 4.6 and pH 5.0 was pH 4.56±0.13 (range of pH 4.20 to pH 4.86) and pH 4.96±0.12 (range of pH 4.70 to pH 5.21), respectively. Fermentation alone delivered ca. a 0.3- to 1.2-log CFU/g reduction in pathogen numbers. Fermentation to ca. pH 4.6 or ca. pH 5.0 followed by post-fermentation heating to 37.8° to 54.4°C and holding for 0.5 to 12.5 h generated total reductions of ca. 2.0 to 6.7 log CFU/g

Chemical Approaches To Perturb, Profile, and Perceive Glycans

Glycosylation is an essential form of post-translational modification that regulates intracellular and extracellular processes. Regrettably, conventional biochemical and genetic methods often fall short for the study of glycans, because their structures are often not precisely defined at the genetic level. To address this deficiency, chemists have developed technologies to perturb glycan biosynthesis, profile their presentation at the systems level, and perceive their spatial distribution. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. Still, glycosylation remains the underexplored frontier of many biological systems. In this Account, we focus on research in our laboratory that seeks to transform the study of glycan function from a challenge to routine practice