Genomic-wide analysis of Salmonella enterica strains isolated from peanuts in Brazil.

Peanut-based products have been associated with Salmonella foodborne outbreaks and/or recalls worldwide. The ability of Salmonella to persist for a long time in a low moisture environment can contribute to this kind of contamination. The objective of this study was to analyse the genome of five S. enterica enterica strains isolated from the peanut supply chain in Brazil, as well as to identify genetic determinants for survival under desiccation and validate these findings by phenotypic test of desiccation stress. The strains were in silico serotyped using the platform SeqSero2 as Miami (M2851), Javiana (M2973), Oranienburg (M2976), Muenster (M624), and Glostrup/Chomedey (M7864); with phylogenomic analysis support. Based on Multilocus Sequence Typing (MLST) the strains were assigned to STs 140, 1674, 321, 174, and 2519. In addition, eight pathogenicity islands were found in all the genomes using the SPIFinder 2.0 (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, SPI-13, SPI-14). The absence of a SPI-4 may indicate a loss of this island in the surveyed genomes. For the pangenomic analysis, 49 S. enterica genomes were input into the Roary pipeline. The majority of the stress related genes were considered as soft-core genes and were located on the chromosome. A desiccation stress phenotypic test was performed in trypticase soy broth (TSB) with four different water activity (aw) values. M2976 and M7864, both isolated from the peanut samples with the lowest aw, showed the highest OD570nm in TSB aw 0.964 and were statistically different (p < 0.05) from the strain isolated from the peanut sample with the highest aw (0.997). In conclusion, genome analyses have revealed signatures of desiccation adaptation in Salmonella strains, but phenotypic analyses suggested the environment influences the adaptive ability of Salmonella to overcome desiccation stress.

Interaction of Aspergillus flavus and A. parasiticus with Salmonella spp. isolated from peanuts.

Although Aspergillus flavus and Aspergillus parasiticus are the main microorganisms of concern in peanuts, due to aflatoxin contamination, several Salmonella outbreaks from this product have been reported over the last ten decades. Thus, it is important to understand the relationship between microorganisms to predict, manage and estimate the diversity in the peanut supply chain. The purpose of this study was to evaluate aflatoxin production during the co-cultivation of Aspergillus section Flavi and Salmonella both isolated from peanuts. Three strains of A. section Flavi: A. flavus producing aflatoxin B, A. flavus non-producing aflatoxin and A. parasiticus producing aflatoxin B and G were co-cultivated with seven serotypes of Salmonella of which six were isolated from the peanut supply chain (S. Muenster, S. Miami, S. Glostrup, S. Javiana, S. Oranienburg and S. Yoruba) and one was S. Typhimurium ATCC 14028. First of all, each Salmonella strain was inoculated by pour plate (ca. 5 log cfu/mL) in PDA (potato dextrose agar). Then, each pre-cultured fungus was inoculated in the center of the petri dish. The plates were incubated at 30 °C and the fungal colony diameter was measured once a day for 7 days. As a control each Aspergillus strain was cultivated in the absence of Salmonella culture. All three strains of Aspergillus with absence of Salmonella (control) reached the maximum colony diameter and their growth rate was influenced when co-cultivated (p < 0.05) with all Salmonella serotypes tested. The maximum inhibition in the colony diameter was 20% for A. flavus aflatoxin B producer and A. parasiticus, and 18% for A. flavus non- aflatoxin producer when cultivated with Salmonella. However, no significant difference (p < 0.05) in reduction of colony diameter was observed among the Salmonella serotypes. Aflatoxin production was determined previously, by using the agar plug technique on thin layer chromatography (TLC). The production of aflatoxin G by A. parasiticus in co-cultivation with Salmonella was not observed. On the other hand, A. flavus preserved their characteristics of aflatoxin B production. The quantification of aflatoxin reduction by Salmonella interaction was evaluated using HPLC method. There was a maximum reduction of aflatoxin production of 88.7% and 72.9% in A. flavus and A. parasiticus, respectively, when cultivated with Salmonella. These results indicate that some serotypes of Salmonella may interfere with aflatoxin production and fungal growth of A. flavus and A. parasiticus in the peanut supply chain.

Genetic diversity, antimicrobial resistance and virulence profile of Salmonella isolated from the peanut supply chain.

Thirty-Eight Salmonella isolates recovered from different stages of the peanut supply chain in three Brazilian States (São Paulo, Minas Gerais and Bahia) were subtyped by pulsed-field gel electrophoresis (PFGE) and characterized by phenotypic and genotypic tests for antimicrobial resistance and virulence genes. The isolates were distributed into seven PFGE pulsotypes. All the isolates were resistant to sulfonamide. However, only one isolate from a production site in Minas Gerais had resistance to two types of antimicrobials (sulfonamide and ampicillin). Furthermore, the isolates had intermediary resistance to kanamycin (16/38), streptomycin (14/38) and ceftazidime (12/38). Four isolates had the antimicrobial resistance gene related to phenicols (floR) and 37 related to aminoglycosides (strA). The blashv gene related to ß-lactams was detected in isolates recovered from all the production regions. Six virulence genes (invA, sefA, sivH, mgtC, ssaQ and agfA) were observed in all isolates. The sopE gene was detected in 24 isolates, avrA in 12. The gtgB, ipfA and rck genes were not detected. The results showed that the pulsotype 1 was restricted to Minas Gerais whereas the pulsotype 7 was present in São Paulo and Bahia. In addition, most of the isolates were not multidrug resistant.

Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee.

Some species from Aspergillus section Nigri are morphologically very similar and altogether have been called A. niger aggregate. Although the species included in this group are morphologically very similar, they differ in their ability to produce mycotoxins and other metabolites and their taxonomical status has evolved continuously. Among them, A. niger and A. welwitschiae are ochratoxin A and fumonisin B2 producers and their detection and/or identification is of crucial importance for food safety. The aim of this study was the development of a real-time PCR-based method for simultaneous discrimination of A. niger and A. welwitschiae from other species of the A. niger aggregate isolated from coffee beans. One primer pair and a hybridization probe specific for detection of A. niger and A. welwitschiae strains were designed based on the BenA gene sequences, and used in a Real-time PCR assay for the rapid discrimination between both these species from all others of the A. niger aggregate. The Real-time PCR assay was shown to be 100% efficient in discriminating the 73 isolates of A. niger/A. welwitschiae from the other A. niger aggregate species analyzed as a negative control. This result testifies to the use of this technique as a good tool in the rapid detection of these important toxigenic species.