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S. enterica isolates (n = 78) obtained from the vegetable supply chain (farms, distribution centers, markets) in two Cambodian provinces (Siem Reap, Battambang) were sequenced and analyzed. In silico identification of serotypes and detection of antimicrobial resistance genes was performed using SISTR and ABRicate, respectively. Isolates’ relatedness was assessed based on high-quality single nucleotide polymorphisms (hqSNPs) identified within each serotype using the CFSAN SNP pipeline. Among 29 detected serotypes, Paratyphi B var. Java was most abundant (n = 14), followed by Hvittingfoss (n = 11) and Thompson (n = 7). Paratyphi B var. Java was mostly found in farms (n = 5) and markets (n = 6), Hvittingfoss within distribution centers (n = 8), and Thompson at markets (n = 4) and farms (n = 3). Among Paratyphi B var. Java isolates, one phylogenetic clade contained four closely related isolates (0–1 SNP difference), collected at markets in different provinces on different days. Another clade contained two isolates that differed by one SNP, one obtained from a Battambang farm and one from a Siem Reap distribution center, suggesting a broad spread of Paratyphi B var. Java in the Cambodian vegetable supply chain. Hvittingfoss isolates clustered in two clades; one contained five identical isolates, four of which were obtained in different months from the distribution center and a farm in Battambang, suggesting possible transmission among supply chain stages. The second clade contained three isolates from the Battambang distribution center that differed by 0–1 SNP and were isolated in October and November, indicating possible persistence. Lastly, among 78 analyzed isolates, 14 carried antimicrobial resistance genes and seven out of these 14 carried genes with predicted resistance to more than three classes of antibiotics. Overall, highly similar isolates of Salmonella were identified over time and at different supply chain stages, suggesting possible persistence and transmission of Salmonella within and between supply chain stages.

Abstract Listeriosis caused by Listeria monocytogenes often poses a significant threat to vulnerable populations. Dairy products have been implicated in outbreaks of listeriosis worldwide. In Ethiopia, studies have identified Listeria spp. and L. monocytogenes in various dairy products, but the genetic diversity and phylogenetic relationships of these bacteria remain largely unknown in the low- and middle-income countries. Therefore, we conducted whole-genome sequencing on 15 L. monocytogenes and 55 L. innocua isolates obtained from different levels of the dairy supply chains across three regions in Ethiopia. Genomes were assembled and used for MLST genotyping and single nucleotide polymorphism (SNP) analysis to infer phylogenetic relationships. We identified a total of 3 L. monocytogenes (i.e., 2, 145, and 18) and 12 L. innocua (i.e., 1489, 1619, 603, 537, 1010, 3186, 492, 3007, 1087, 474, 1008, and 637) MLST sequence types among the studied isolates. Some of these sequence types showed region-specific occurrence, while others were broadly distributed across regions. Through high-quality SNP analysis, we found that among 13 L. monocytogenes identified as ST 2, 11 of them were highly similar with low genetic variation, differing by only 1 to 10 SNPs, suggesting potential selection in the dairy food supply chain. The L. innocua isolates also exhibited low intra-ST genetic variation with only 0–10 SNP differences, except for the ST 1619, which displayed a greater diversity.

A major concern for plant nurseries and land stewards is managing pathogens that induce soilborne diseases. Plant diseases cost nurseries millions of dollars in lost revenue and plant pathogens spread into the environment from nursery soil used for out-plantings. Recent research shows that steaming soil at high temperatures can effectively kill plant pathogens; however, few studies have investigated the influence of steaming on the soil microbiome, an essential component of plant health. We conducted a controlled experiment where nursery soil underwent a steaming event within a rectangular mesocosm (2.04 × 3.55 × 0.25 m) located at the National Ornamentals Research Site at Dominican University (NORS-DUC) (San Rafael, CA, U.S.A.). Soil samples were collected at four time points post-steaming. Thirty-two soil samples were used for DNA extraction and 18 for chemical analysis. The 16S rRNA gene and ITS genetic markers were used for amplicon-based sequencing to identify the bacterial and fungal soil communities. Results indicate that microbial diversity and composition shifted over time but that soil depth was not an important factor. Important bacterial taxa were the Firmicutes, especially 1 day post-steaming, and common fungal taxa included Aspergillus sp., Cladosporium sp., and Pseudallescheria sp. Plant nutrients such as B, C, Cl, K, Mg, SO4, and Zn, in addition to essential soil properties such as CEC, gypsum, moisture, and SAR, changed significantly with soil recovery. These results can help nursery practitioners make informed decisions for biotic and nutrient amendments post-steaming to promote soil and plant health. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Background Listeria monocytogenes can survive in cold and wet environments, such as tree fruit packing facilities and it has been implicated in outbreaks and recalls of tree fruit products. However, little is known about microbiota that co-occurs with L. monocytogenes and its stability over seasons in tree fruit packing environments. In this 2-year longitudinal study, we aimed to characterize spatial and seasonal changes in microbiota composition and identify taxa indicative of L. monocytogenes contamination in wet processing areas of three tree fruit packing facilities (F1, F2, F3). Methods A total of 189 samples were collected during two apple packing seasons from floors under the washing, drying, and waxing areas. The presence of L. monocytogenes was determined using a standard culturing method, and environmental microbiota was characterized using amplicon sequencing. PERMANOVA was used to compare microbiota composition among facilities over two seasons, and abundance-occupancy analysis was used to identify shared and temporal core microbiota. Differential abundance analysis and random forest were applied to detect taxa indicative of L. monocytogenes contamination. Lastly, three L. monocytogenes-positive samples were sequenced using shotgun metagenomics with Nanopore MinION, as a proof-of-concept for direct detection of L. monocytogenes’ DNA in environmental samples. Results The occurrence of L. monocytogenes significantly increased from 28% in year 1 to 46% in year 2 in F1, and from 41% in year 1 to 92% in year 2 in F3, while all samples collected from F2 were L. monocytogenes-positive in both years. Samples collected from three facilities had a significantly different microbiota composition in both years, but the composition of each facility changed over years. A subset of bacterial taxa including Pseudomonas, Stenotrophomonas, and Microbacterium, and fungal taxa, including Yarrowia, Kurtzmaniella, Cystobasidium, Paraphoma, and Cutaneotrichosporon, were identified as potential indicators of L. monocytogenes within the monitored environments. Lastly, the DNA of L. monocytogenes was detected through direct Nanopore sequencing of metagenomic DNA extracted from environmental samples. Conclusions This study demonstrated that a cross-sectional sampling strategy may not accurately reflect the representative microbiota of food processing facilities. Our findings also suggest that specific microorganisms are indicative of L. monocytogenes, warranting further investigation of their role in the survival and persistence of L. monocytogenes. Video Abstract

Abstract Background Multistate foodborne disease outbreaks and recalls of apples and apple products contaminated with Listeria monocytogenes demonstrate the need for improved pathogen control in the apple supply chain. Apple processing facilities have been identified in the past as potential sources of persisting L. monocytogenes contamination. In this study, we sought to understand the composition of microbiota in built apple and other tree fruit processing environments and its association with the occurrence of the foodborne pathogen L. monocytogenes. Results Analysis of 117 samples collected from three apple and other tree fruit packing facilities (F1, F2, and F3) showed that facility F2 had a significantly higher L. monocytogenes occurrence compared to F1 and F3 (p < 0.01). The microbiota in facility F2 was distinct compared to facilities F1 and F3 as supported by the mean Shannon index for bacterial and fungal alpha diversities that was significantly lower in F2, compared to F1 and F3 (p < 0.01). Microbiota in F2 was uniquely predominated by bacterial family Pseudomonadaceae and fungal family Dipodascaceae. Conclusions The composition and diversity of microbiota and mycobiota present in the investigated built food processing environments may be indicative of persistent contamination with L. monocytogenes. These findings support the need for further investigation of the role of the microbial communities in the persistence of L. monocytogenes to support the optimization of L. monocytogenes control strategies in the apple supply chain.

This white paper presents a series of perspectives on current and future phytobiome management, discussed at the Wild and Tamed Phytobiomes Symposium in University Park, PA, U.S.A., in June 2018. To enhance plant productivity and health, and to translate lab- and greenhouse-based phytobiome research to field applications, the academic community and end-users need to address a variety of scientific, practical, and social challenges. Prior discussion of phytobiomes has focused heavily on plant-associated bacterial and fungal assemblages, but the phytobiomes concept covers all factors that influence plant function. Here we discuss various management considerations, including abiotic conditions (e.g., soil and nutrient applications), microorganisms (e.g., bacterial and fungal assemblages, bacterial and fungal inoculants, and viruses), macroorganisms (e.g., arthropods and plant genetics), and societal factors (e.g., communication approaches and technology diffusion). An important near-term goal for this field should be to estimate the potential relative contribution of different components of the phytobiome to plant health, as well as the potential and risk of modifying each in the near future.

Surface water used for produce production is a potential source of pre-harvest contamination with foodborne pathogens. Decisions on how to mitigate food safety risks associated with pre-harvest water use currently rely on generic Escherichia coli-based water quality tests, although multiple studies have suggested that E. coli levels are not a suitable indicator of the food safety risks under all relevant environmental conditions. Hence, improved understanding of spatiotemporal variability in surface water microbiota composition is needed to facilitate identification of alternative or supplementary indicators that co-occur with pathogens. To this end, we aimed to characterize the composition of bacterial and fungal communities in the sediment and water fractions of 68 agricultural water samples collected from six New York streams. We investigated potential associations between the composition of microbial communities, environmental factors and Salmonella and/or Listeria monocytogenes isolation. We found significantly different composition of fungal and bacterial communities among sampled streams and among water fractions of collected samples. This indicates that geography and the amount of sediment in a collected water sample may affect its microbial composition, which was further supported by identified associations between the flow rate, turbidity, pH and conductivity, and microbial community composition. Lastly, we identified specific microbial families that were weakly associated with the presence of Salmonella or Listeria monocytogenes, however, further studies on samples from additional streams are needed to assess whether identified families may be used as indicators of pathogen presence.

Abstract Background Malonate utilization, an important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak). The current study provides experimental evidence that confirms the presence of a malonate utilization operon in 24 strains of sequence type (ST) 64, obtained from Europe, Middle East, China, and USA; it offers explanations regarding the genomic diversity and phylogenetic relatedness among these strains, and that of other C. sakazakii strains. Results In this study, the presence of a malonate utilization operon in these strains was initially identified by DNA microarray analysis (MA) out of a pool of 347 strains obtained from various surveillance studies involving clinical, spices, milk powder sources and powdered infant formula production facilities in Ireland and Germany, and dried dairy powder manufacturing facilities in the USA. All ST64 C. sakazakii strains tested could utilize malonate. Zebrafish embryo infection studies showed that C. sakazakii ST64 strains are as virulent as other Cronobacter species. Parallel whole genome sequencing (WGS) and MA showed that the strains phylogenetically grouped as a separate clade among the Csak species cluster. Additionally, these strains possessed the Csak O:2 serotype. The nine-gene, ~ 7.7 kbp malonate utilization operon was located in these strains between two conserved flanking genes, gyrB and katG. Plasmidotyping results showed that these strains possessed the virulence plasmid pESA3, but in contrast to the USA ST64 Csak strains, ST64 Csak strains isolated from sources in Europe and the Middle East, did not possess the type six secretion system effector vgrG gene. Conclusions Until this investigation, the presence of malonate-positive Csak strains, which are associated with foods and clinical cases, was under appreciated. If this trait was used solely to identify Cronobacter strains, many strains would likely be misidentified. Parallel WGS and MA were useful in characterizing the total genome content of these Csak O:2, ST64, malonate-positive strains and further provides an understanding of their phylogenetic relatedness among other virulent C. sakazakii strains.

The Bacillus cereus sensu stricto (s.s.) species comprises strains of biovar Thuringiensis (Bt) known for their bioinsecticidal activity, as well as strains with foodborne pathogenic potential. Bt strains are identified (i) based on the production of insecticidal crystal proteins, also known as Bt toxins, or (ii) based on the presence of cry, cyt, and vip genes, which encode Bt toxins. Multiple bioinformatics tools have been developed for the detection of crystal protein-encoding genes based on wholegenome sequencing (WGS) data. However, the performance of these tools is yet to be evaluated using phenotypic data. Thus, the goal of this study was to assess the performance of four bioinformatics tools for the detection of crystal protein-encoding genes. The accuracy of sequence-based identification of Bt was determined in reference to phenotypic microscope-based screening for the production of crystal proteins. A total of 58 diverse B. cereus sensu lato strains isolated from clinical, food, environmental, and commercial biopesticide products underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS (identification of pesticidal sequences), and Cry_processor. Out of 58 isolates, the phenotypic production of crystal proteins was confirmed for 18 isolates. Specificity and sensitivity of Bt ident ification based on sequences were 0.85 and 0.94 for BtToxin_Digger, 0.97 and 0.89 for BTyper3, 0.95 and 0.94 for IDOPS, and 0.88 and 1.00 for Cry_processor, respectively. Cry_processor predicted crystal protein production with the highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatics tools performed well overall, with IDOPS achieving high sensitivity and specificity (>0.90). IMPORTANCE Strains of Bacillus cereus sensu stricto (s.s.) biovar Thuringiensis (Bt) are used as organic biopesticides. Bt is differentiated from the foodborne pathogen Bacillus cereus s.s. by the production of insecticidal crystal proteins. Thus, reliable genomic identification of biovar Thuringiensis is necessary to ensure food safety and facilitate risk assessment. This study assessed the accuracy of whole-genome sequencing (WGS)- based identification of Bt compared to phenotypic microscopy-based screening for crystal protein production. Multiple bioinformatics tools were compared to assess their performance in predicting crystal protein production. Among them, identification of pesticidal sequences performed best overall at WGS-based Bt identification. [ABSTRACT FROM AUTHOR]

Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X;MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport. [ABSTRACT FROM AUTHOR]