Computational assessment of phytochemicals of medicinal plants from Mexico as potential inhibitors of Salmonella enterica efflux pump AcrB protein.

The AcrAB-TolC efflux pump (EP) confers multidrug resistance to Salmonella enterica, a major etiological agent of foodborne infections. Phytochemicals that inhibit the functions of AcrAB-TolC EP present ideal candidates for reversal of antibiotic resistance. Progressive technological advancements, have facilitated the development of computational methods that offer a rapid low-cost approach to screen and identify phytochemicals with inhibitory potential against EP. In this study, 71 phytochemicals derived from plants used for medicinal purposes in Mexico were screened for their potential as inhibitors of Salmonella AcrB protein using in silico approaches including molecular docking and molecular dynamics (MD) simulation. Consequently, naringenin, 5-methoxypsoralen, and licarin A were identified as candidate inhibitors of AcrB protein. The three phytochemicals bound distal/deep pocket (DP) and hydrophobic trap (HPT) residues of AcrB protein critical for interactions with inhibitors, with estimated binding free energies of -95.5 kJ/mol, -97.4 kJ/mol, and -143.8 kJ/mol for naringenin, 5-methoxypsoralen, and licarin A, respectively. Data from the 50 ns MD simulation study revealed stability of the protein-ligand complex and alterations in the AcrB protein DP conformation upon binding of phytochemicals to the DP and HPT regions. Based on the estimated binding free energy and interactions with three out of five residues lining the hydrophobic trap, licarin A demonstrated the highest inhibitory potential, supporting its further application as a candidate for overcoming drug resistance in pathogens. Communicated by Ramaswamy H. Sarma.

Epigallocatechin gallate and Lactobacillus plantarum culture supernatants exert bactericidal activity and reduce biofilm formation in Clostridium perfringens.

Clostridium perfringens forms biofilms and spores that are a source of food contamination. In this study, the antibacterial activities of Lactobacillus plantarum culture supernatants (LP-S), LP-S fractions, and the plant-derived compound epigallocatechin gallate (EG) were evaluated. Specifically, their effects on the viability and biofilm-forming ability of C. perfringens were assessed. Moreover, the expression of quorum sensing-regulated genes associated with the pathogenesis of this microorganism and that of genes involved in biofilm formation was also investigated. The results showed that both EG and the LP-S exerted bactericidal activity against all C. perfringens strains tested. The minimal bactericidal concentration (MBC) of EG was 75 µg/mL for all strains but ranged from 61 to 121 µg of total protein per mL for LP-S. EG exerted only minor effects on biofilm formation, whereas LP-S, particularly its 10 and 30 K fractions, significantly reduced the biofilm-forming ability of all the strains. The antibiofilm activity of LP-S was lost following preincubation with proteases, suggesting that it was mediated by a proteinaceous molecule. The treatment of C. perfringens with either EG or LP-S did not change the transcript levels of two CpAL (C. perfringens quorum-sensing Agr-like system)-related genes, agrB and agrD, which are known to be involved in the regulation of biofilms, suggesting that LP-S exerted its biofilm inhibitory activity downstream of CpAL signaling. In summary, we demonstrated the bactericidal activity of EG and LP-S against C. perfringens and antibiofilm activity of LP-S at a subinhibitory dose. Our results suggested that these compounds can be further explored for food safety applications to control agents such as C. perfringens.

The Cantaloupe Farm Environment Has a Diverse Genetic Pool of Antibiotic-Resistance and Virulence Genes.

Cantaloupes contaminated with pathogens have led to many high-profile outbreaks and illnesses. Since bacterial virulence genes (VGs) can act in tandem with antibiotic-resistance and mobile genetic elements, there is a need to evaluate these gene reservoirs in fresh produce, such as cantaloupes. The goal of this study was to assess the distribution of antibiotic-resistance, virulence, and mobile genetic elements genes (MGEGs) in cantaloupe farm environments. A total of 200 samples from cantaloupe melons (n = 99), farm workers' hands (n = 66), and production water (n = 35) were collected in México. Each sample was assayed for the presence of 14 antibiotic-resistance genes, 15 VGs, and 5 MGEGs by polymerase chain reaction. Our results indicated that tetracycline (tetA and tetB) (18% of cantaloupe, 45% of hand samples) and sulfonamide (sul1) (30% of cantaloupe, 71% of hand samples) resistance genes were frequently detected. The colistin resistance gene (mcr1) was detected in 10% of cantaloupe and 23% of farm workers' hands. Among VGs, Salmonella genes invA and spiA were the most abundant. There was a significantly higher likelihood of detecting antibiotic-resistance, virulence, and MGEGs on hands compared with water samples. These results demonstrate a diverse pool of antibiotic-resistance and VGs in cantaloupe production.

Analysis of Bacterial Communities by 16S rRNA Gene Sequencing in a Melon-Producing Agro-environment.

Cantaloupe melons, which have been responsible of an increasing number of foodborne disease outbreaks, may become contaminated with microbial pathogens during production. However, little information is available on the microbial populations in the cantaloupe farm environment. The purpose of this work was to characterize the bacterial communities present on cantaloupe farms. Fruit, soil, and harvester hand rinsates were collected from two Mexican cantaloupe farms, each visited three times. Microbiome analysis was performed by sequencing 16sRNA and analyzed using qiime2 software. Correlations were determined between sample type and microbial populations. The α and ß diversity analysis identified 2777 sequences across all samples. The soil samples had the highest number and diversity of unique species (from 130 to 1329 OTUs); cantaloupe (from 112 to 205 OTUs), and hands (from 67 to 151 OTUs) had similar diversity. Collectively, Proteobacteria was the most abundant phyla (from 42 to 95%), followed by Firmicutes (1-47%), Actinobacteria (< 1 to 23%), and Bacteroidetes (< 1 to 4.8%). The most abundant genera were Acinetobacter (20-58%), Pseudomonas (14.5%), Erwinia (13%), and Exiguobacterium (6.3%). Genera with potential to be pathogenic included Bacillus (4%), Salmonella (0.85%), Escherichia-Shigella (0.38%), Staphylococcus (0.32%), Listeria (0.29%), Clostridium (0.28%), and Cronobacter (0.27%), which were found at lower frequencies. This study provides information on the cantaloupe production microbiome, which can inform future research into critical food safety issues such as antimicrobial resistance, virulence, and genomic epidemiology.

Transcriptomic analysis of the role of Rim101/PacC in the adaptation of Ustilago maydis to an alkaline environment.

Alkaline pH triggers an adaptation mechanism in fungi that is mediated by Rim101/PacCp, a zinc finger transcription factor. To identify the genes under its control in Ustilago maydis, we performed microarray analyses, comparing gene expression in a wild-type strain versus a rim101/pacC mutation strain of the fungus. In this study we obtained evidence of the large number of genes regulated mostly directly, but also indirectly (probably through regulation of other transcription factors), by Rim101/PacCp, including proteins involved in a large number of physiological activities of the fungus. Our analyses suggest that the response to alkaline conditions under the control of the Pal/Rim pathway involves changes in the cell wall and plasma membrane through alterations in their lipid, protein and polysaccharide composition, changes in cell polarity, actin cytoskeleton organization, and budding patterns. Also as expected, adaptation involves regulation by Rim101/PacC of genes involved in meiotic functions, such as recombination and segregation, and expression of genes involved in ion and nutrient transport, as well as general vacuole functions.