Study of an Enterococcus faecium strain isolated from an artisanal Mexican cheese, whole-genome sequencing, comparative genomics, and bacteriocin expression.

Enterococci are ubiquitous microorganisms in almost all environments, from the soil we step on to the food we eat. They are frequently found in naturally fermented foods, contributing to ripening through protein, lipid, and sugar metabolism. On the other hand, these organisms are also leading the current antibiotic resistance crisis. In this study, we performed whole-genome sequencing and comparative genomics of an Enterococcus faecium strain isolated from an artisanal Mexican Cotija cheese, namely QD-2. We found clear genomic differences between commensal and pathogenic strains, particularly in their carbohydrate metabolic pathways, resistance to vancomycin and other antibiotics, bacteriocin production, and bacteriophage and CRISPR content. Furthermore, a bacteriocin transcription analysis performed by RT-qPCR revealed that, at the end of the log phase, besides enterocins A and X, two putative bacteriocins not reported previously are also transcribed as a bicistronic operon in E. faecium QD-2, and are expressed 1.5 times higher than enterocin A when cultured in MRS broth.

Microbiological Evaluation of Two Mexican Artisanal Cheeses: Analysis of Foodborne Pathogenic Bacteria in Cotija Cheese and Bola de Ocosingo Cheese by qPCR.

Cotija and Bola de Ocosingo are artisanal ripened cheeses produced in Mexico. Both are made with raw bovine milk from free-grazing cows and with no starter cultures. Unlike culture-based techniques, molecular methods for pathogen detection in food allow a shorter turnaround time, higher detection specificity, and represent a lower microbiological risk for the analyst. In the present investigation, we analyzed 111 cheese samples (95 Cotija and 16 Bola de Ocosingo) by qPCR (TaqMan®) after an enrichment-culture step specific to each foodborne bacterium. The results showed that 100% of the samples were free of DNA from Listeria monocytogenes, Brucella spp., Escherichia coli enterotoxigenic (ETEC), and O157:H7; 9% amplified Salmonella spp. DNA; and 11.7%, Staphylococcus aureus DNA. However, the threshold cycle (Ct) values of the amplified targets ranged between 23 and 30, indicating DNA from non-viable microorganisms. Plate counts supported this assumption. In conclusion, 100% of the cheeses analyzed were safe to consume, and the enrichment step before DNA extraction proved essential to discern between viable and non-viable microorganisms. Hygienic milking, milk handling, cheese manufacturing, and ripening are crucial to achieve an adequate microbiological quality of cheeses made with raw milk.

Real-time and conventional PCR detection of Liberty Link rice varieties and transgenic soy in rice sampled in the Mexican and American retail markets.

Samples of rice from Mexican and USA retail stores were analyzed for the presence of transgenic (GM) events using real-time PCR. In screening for the CaMV35S promoter sequence (35SP), positive results were found in 49 and 35% of the Mexican and American samples, respectively. In further investigations in Mexican samples, 43% were positive for P35S::bar, with two above the quantifiable limit; these were 0.07% and 0.05% GMO. Fourteen out of the sixteen positive samples were labeled as imported from the USA. In testing samples bought in American retail shops, 24% showed positive results, all below the quantifiable range. It could be deduced that P35S::bar positive samples were Liberty Link(R) (LL) rice. In distinguishing between LL601 and LL62, end-point PCR was used, corroborating the P35S::bar amplicon length difference of these events. LL62 was found in one rice sample purchased in Mexico and two in the USA samples. Its presence was verified with the 35S terminator sequence. All other LL positive samples contained LL601. None of the samples analyzed showed the presence of Bt63 rice. The LL rice varieties found have been identified as not being commercially cultivated, and so their presence requires further investigation. 35SP was also present in samples which did not have any LL rice. Maize sequences could not be detected in any of the samples; however, soybean DNA was found in Mexican and USA rice samples. The Roundup Ready(R) trait was detected in trace amounts in 16 and 6% of the rice samples bought in Mexico and the USA, respectively. Real-time PCR was shown to be the method of choice for the sensitive and rapid screening of commodities and retail samples for the detection of GM and other contamination.

Enhancement of the antibacterial activity of an E. faecalis strain by the heterologous expression of enterocin A.

The genus Enterococcus occurs as native microbiota of fermented products due to its broad environmental distribution and its resistance to salt concentrations. Enterococcus faecalis F, a non-pathogenic strain isolated from a ripened cheese, has demonstrated useful enzymatic capabilities, a probiotic behavior and antibacterial activity against some food-borne pathogens, mainly due to peptidoglycan hydrolase activity. Its use as a natural pathogen-control agent could be further enhanced through the production of a bacteriocin, e.g. Enterocin A, because of its remarkable antilisterial activity. In this work, a markerless allelic insertion method was used to obtain an enterococcal strain capable of producing a functional enterocin. Agar diffusion tests showed that the recombinant strain was active against Staphylococcus aureus, Listeria monocytogenes and the pathogenic strain E. faecalis V583. When grown in liquid culture together with L. monocytogenes, it attained a two-log reduction of the pathogen counts in lesser time relative to the native strain. Because the DNA construction is integrated into the chromosome, the improved strain avoids the use of antibiotics as selective pressure; besides, it does not require an inductor because of the inclusion of a constitutive promoter in the construction. Its technological and antibacterial capabilities make the improved E. faecalis strain a potential culture for use in the food industry.

Cloning and Characterization of a Novel N-acetylglucosaminidase (AtlD) from Enterococcus faecalis.

The atlD gene from an Enterococcus faecalis strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in Escherichia coli in order to perform a biochemical characterization. A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62-75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6-7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn2+. It showed antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and enterococcal strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.

Characterization of a 19 kDa alpha-zein of high purity.

A highly pure alpha-zein was extracted from corn flour using ethanol (95%). Subsequently, ion-exchange chromatography was performed, using SP-Sepharose that yielded a highly homogeneous protein. This protein migrated as a single band in 20% SDS-PAGE and in pH gradient gels, showing an isoelectric point of 6.8. Mass spectrometry (MALDI-TOF-MS) showed a single peak with a molecular mass of 24 535 Da. It was identified as Z19, when comparing the sequence obtained in an automatic Edman sequencer with the Swissprot database using BLAST. The molar extinction coefficient, determined by dry weight in 70% methanol, was 12 415.49 M(-1) cm(-1) at 280 nm. Light scattering showed its presence in a monodispersed state of 44-66 kDa aggregates in methanol (70%). Circular dichroism spectra allowed the estimation of an alpha-helix content that was lower than the one found for a mixture of two alpha-zeins but with a higher content of beta sheets.

Suitability of real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay for cry9C detection in Mexican corn tortillas: fate of DNA and protein after alkaline cooking.

Alkaline-cooked corn, called nixtamal, is the basis for many traditional corn products such as tortillas, chips, and taco shells that are used widely in Mexico and Central America and in the preparation of snack foods that are consumed globally. To assess the effects of alkaline and thermal treatments on the detectability of DNA and protein for the presence of genetically modified sequences, various nixtamalized products were prepared from blends of conventional white corn containing 0.1, 1.0, and 10% transgenic corn (event CBH 351, StarLink). Real-time quantitative polymerase chain reactions (RTQ-PCR) and immunoassays were used to determine the cry9C gene and protein, respectively, in unprocessed corn kernels, freshly prepared alkaline-cooked and ground corn (masa), masa flour, tortillas prepared from masa by heat treatment, chips prepared from damp masa dough by deep frying, and from tortillas processed at high (200 degrees C) and low temperatures (70 degrees C). In spite of progressive degradation of genomic DNA during processing, RTQ-PCR genetic analysis allowed detection and quantification of the cry9C gene in all products prepared from 10, 1, and 0.1% StarLink corn, except deep-fried chips containing 0.1% StarLink. Enzyme-linked immunosorbent assays readily detected <1 ppm cry9C protein in all blends of unprocessed corn (10, 1, and 0.1% StarLink) as well as in nonfried tortilla and masa products. This technique was not suitable for thermally treated nixtamalized products containing <1% transgenic corn.

Proteolytic modification of Leuconostoc mesenteroides B-512F dextransucrase.

Multiple active lower molecular weight forms from Leuconostoc mesenteroides B512F dextransucrase have been reported. It has been suggested that they arise from proteolytic processing of a 170 kDa precursor. In this work, the simultaneous production of proteases and dextransucrase was studied in order to elucidate the dextransucrase proteolytic processing. The effect of the nitrogen source on protease and dextransucrase production was studied. Protease activity reaches a maximum early in the logarithmic phase of dextransucrase synthesis using the basal culture medium but the nitrogen source plays an important effect on growth: the highest protease concentration was obtained when ammonium sulfate, casaminoacids or tryptone were used. Two active forms of 155 and 129 kDa were systematically obtained from dextransucrase precursor by proteolysis. The amino termini of these forms were sequenced and the cleavage site deduced. Both forms of the enzyme obtained had the same cleavage site in the amino terminal region (F209-Y210). From dextransucrase analysis, various putative cleavage sites with the same sequence were found in the variable region and in the glucan binding domain. Although no structural differences were found in dextrans synthesized with both the precursor and the proteolyzed 155 kDa form under the same reaction conditions, their rheological behaviour was modified, with dextran of a lower viscosity yielded by the smaller form.