Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States.

The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of ≥ 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers' health.

Bacterial community shifts of commercial apples, oranges, and peaches at different harvest points across multiple growing seasons.

Assessing the microbes present on tree fruit carpospheres as the fruit enters postharvest processing could have useful applications, as these microbes could have a major influence on spoilage, food safety, verification of packing process controls, or other aspects of processing. The goal of this study was to establish a baseline profile of bacterial communities associated with apple (pome fruit), peach (stone fruit), and Navel orange (citrus fruit) at harvest. We found that commercial peaches had the greatest bacterial richness followed by oranges then apples. Time of harvest significantly changed bacterial diversity in oranges and peaches, but not apples. Shifts in diversity varied by fruit type, where 70% of the variability in beta diversity on the apple carposphere was driven by the gain and loss of species (i.e., nestedness). The peach and orange carposphere bacterial community shifts were driven by nearly an even split between turnover (species replacement) and nestedness. We identified a small core microbiome for apples across and between growing seasons that included only Methylobacteriaceae and Sphingomonadaceae among the samples, while peaches had a larger core microbiome composed of five bacterial families: Bacillaceae, Geodermtophilaceae, Nocardioidaceae, Micrococcaeceae, and Trueperaceae. There was a relatively diverse core microbiome for oranges that shared all the families present on apples and peaches, except for Trueperaceae, but also included an additional nine bacterial families not shared including Oxalobacteraceae, Cytophagaceae, and Comamonadaceae. Overall, our findings illustrate the important temporal dynamics of bacterial communities found on major commercial tree fruit, but also the core bacterial families that constantly remain with both implications being important entering postharvest packing and processing.

Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool.

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.

Genomic Analysis Points to Multiple Genetic Mechanisms for Non-Transformable Campylobacter jejuni ST-50.

Campylobacter jejuni and Campylobacter coli are well known for their natural competence, i.e., their capacity for the uptake of naked DNA with subsequent transformation. This study identifies non-transformable C. jejuni and C. coli strains from domestic animals and employs genomic analysis to investigate the strain genotypes and their associated genetic mechanisms. The results reveal genetic associations leading to a non-transformable state, including functional DNase genes from bacteriophages and mutations within the cts-encoded DNA-uptake system, which impact the initial steps of the DNA uptake during natural transformation. Interestingly, all 38 tested C. jejuni ST-50 strains from the United States exhibit a high prevalence of non-transformability, and the strains harbor a variety of these genetic markers. This research emphasizes the role of these genetic markers in hindering the transfer of antimicrobial resistance (AMR) determinants, providing valuable insights into the genetic diversity of Campylobacter. As ST-50 is a major clone of C. jejuni globally, we additionally determined the prevalence of the genetic markers for non-transformability among C. jejuni ST-50 from different regions of the world, revealing distinct patterns of evolution and a strong selective pressure on the loss of competence in ST-50 strains, particularly in the agricultural environment in the United States. Our findings contribute to a comprehensive understanding of genetic exchange mechanisms within Campylobacter strains, and their implications for antimicrobial resistance dissemination and evolutionary pathways within specific lineages.

Genomic resistant determinants of multidrug-resistant Campylobacter spp. isolates in Peru.

OBJECTIVES: Antimicrobial resistant (AMR) Campylobacter is a global health threat; however, there is limited information on genomic determinants of resistance in low- and middle-income countries. We evaluated genomic determinants of AMR using a collection of whole genome sequenced Campylobacter jejuni and C. coli isolates from Iquitos, Peru. METHODS: Campylobacter isolates from two paediatric cohort studies enriched with isolates that demonstrated resistance to ciprofloxacin and azithromycin were sequenced and mined for AMR determinants. RESULTS: The gyrA mutation leading to the Thr86Ile amino acid change was the only gyrA mutation associated with fluoroquinolone resistance identified. The A2075G mutation in 23S rRNA was present, but three other 23S rRNA mutations previously associated with macrolide resistance were not identified. A resistant-enhancing variant of the cmeABC efflux pump genotype (RE-cmeABC) was identified in 36.1% (35/97) of C. jejuni genomes and 17.9% (12/67) of C. coli genomes. Mutations identified in the CmeR-binding site, an inverted repeat sequence in the cmeABC promoter region that increases expression of the operon, were identified in 24/97 C. jejuni and 14/67 C. coli genomes. The presence of these variants, in addition to RE-cmeABC, was noted in 18 of the 24 C. jejuni and 9 of the 14 C. coli genomes. CONCLUSIONS: Both RE-cmeABC and mutations in the CmeR-binding site were strongly associated with the MDR phenotype in C. jejuni and C. coli. This is the first report of RE-cmeABC in Peru and suggests it is a major driver of resistance to the principal therapies used to treat human campylobacteriosis in this setting.

Shotgun metagenomics of fecal samples from children in Peru reveals frequent complex co-infections with multiple Campylobacter species.

Campylobacter spp. are a major cause of bacterial diarrhea worldwide and are associated with high rates of mortality and linear growth faltering in children living in low- to middle-income countries (LMICs). Campylobacter jejuni and Campylobacter coli are most often the causative agents of enteric disease among children in LMICs. However, previous work on a collection of stool samples from children under 2 years of age, living in a low resource community in Peru with either acute diarrheal disease or asymptomatic, were found to be qPCR positive for Campylobacter species but qPCR negative for C. jejuni and C. coli. The goal of this study was to determine if whole-genome shotgun metagenomic sequencing (WSMS) could identify the Campylobacter species within these samples. The Campylobacter species identified in these stool samples included C. jejuni, C. coli, C. upsaliensis, C. concisus, and the potential new species of Campylobacter, "Candidatus Campylobacter infans". Moreover, WSMS results demonstrate that over 65% of the samples represented co-infections with multiple Campylobacter species present in a single stool sample, a novel finding in human populations.

"Candidatus Campylobacter infans" detection is not associated with diarrhea in children under the age of 2 in Peru.

A working hypothesis is that less common species of Campylobacter (other than C. jejuni and C. coli) play a role in enteric disease among children in low resource settings and explain the gap between the detection of Campylobacter using culture and culture independent methods. "Candidatus Campylobacter infans" (C. infans), was recently detected in stool samples from children and hypothesized to play a role in Campylobacter epidemiology in low- and middle-income countries (LMIC). This study determined the prevalence of C. infans in symptomatic and asymptomatic stool samples from children living in Iquitos, Peru. Stool samples from 215 children with diarrhea and 50 stool samples from children without diarrhea under the age of two were evaluated using a multiplex qPCR assay to detect Campylobacter spp. (16S rRNA), Campylobacter jejuni / Campylobacter coli (cadF gene), C. infans (lpxA), and Shigella spp. (ipaH). C. infans was detected in 7.9% (17/215) symptomatic samples and 4.0% (2/50) asymptomatic samples. The association between diarrhea and the presence of these targets was evaluated using univariate logistic regressions. C. infans was not associated with diarrhea. Fifty-one percent (75/146) of Campylobacter positive fecal samples were negative for C. jejuni, C. coli, and C. infans via qPCR. Shotgun metagenomics confirmed the presence of C. infans among 13 out of 14 positive C. infans positive stool samples. C infans explained only 20.7% of the diagnostic gap in stools from children with diarrhea and 16.7% of the gap in children without diarrhea. We posit that poor cadF primer performance better explains the observed gap than the prevalence of atypical non-C. jejuni/coli species.

Complete Genome Sequences of Two Bacillus velezensis Strains Isolated from California Raisin Vineyard Soils.

Bacillus velezensis strains JP3042 and JP3144 were isolated from California raisin vineyard soils and were selected for further study of in vitro antifungal activity. Here, we present the complete genome sequences of these strains to aid in the understanding of their antifungal activity and diversity within the species.

The bile salt deoxycholate induces Campylobacter jejuni genetic point mutations that promote increased antibiotic resistance and fitness.

Oxidative damage to DNA is a significant source of mutations in living organisms. While DNA damage must be repaired to maintain the integrity of the genome and cell survival, errors made during DNA repair may contribute to evolution. Previous work has revealed that Campylobacter jejuni growth in the presence of bile salt deoxycholate (DOC) causes an increase in reactive oxygen species and the occurrence of 8-oxo-deoxyguanosine (8-oxo-dG) DNA lesions. The fundamental goal of this project was to determine if C. jejuni growth in a medium containing DOC contributes to DNA mutations that provide a fitness advantage to the bacterium. Co-culture experiments revealed that C. jejuni growth in a DOC-supplemented medium increases the total number of ciprofloxacin-resistant isolates compared to C. jejuni grown in the absence of DOC. We recovered two individual isolates grown in a medium with DOC that had a point mutation in the gene encoding the EptC phosphoethanolamine transferase. Transformants harboring the EptC variant protein showed enhanced resistance to the antimicrobial agent polymyxin B and DOC when compared to an eptC deletion mutant or the isolate complemented with a wild-type copy of the gene. Finally, we found that the base excision repair (BER), homologous recombination repair (HRR), and nucleotide excision repair (NER) are involved in general oxidative damage repair in C. jejuni but that the BER pathway plays the primary role in the repair of the 8-oxo-dG lesion. We postulate that bile salts drive C. jejuni mutations (adaptations) and enhance bacterial fitness in animals.

Utilization Efficiency of Human Milk Oligosaccharides by Human-Associated Akkermansia Is Strain Dependent.

Akkermansia muciniphila is a mucin-degrading bacterium found in the human gut and is often associated with positive human health. However, despite being detected by as early as 1 month of age, little is known about the role of Akkermansia in the infant gut. Human milk oligosaccharides (HMOs) are abundant components of human milk and are structurally similar to the oligosaccharides that comprise mucin, the preferred growth substrate of human-associated Akkermansia. A limited subset of intestinal bacteria has been shown to grow well on HMOs and mucin. We therefore examined the ability of genomically diverse strains of Akkermansia to grow on HMOs. First, we screened 85 genomes representing the four known Akkermansia phylogroups to examine their metabolic potential to degrade HMOs. Furthermore, we examined the ability of representative isolates to grow on individual HMOs in a mucin background and analyzed the resulting metabolites. All Akkermansia genomes were equipped with an array of glycoside hydrolases associated with HMO deconstruction. Representative strains were all able to grow on HMOs with various efficiencies and growth yields. Strain CSUN-19, belonging to the AmIV phylogroup, grew to the highest level in the presence of fucosylated and sialylated HMOs. This activity may be partially related to the increased copy numbers and/or the enzyme activities of the α-fucosidases, α-sialidases, and ß-galactosidases. This study examines the utilization of individual purified HMOs by Akkermansia strains representing all known phylogroups. Further studies are required to examine how HMO ingestion influences gut microbial ecology in infants harboring different Akkermansia phylogroups. IMPORTANCE Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk and provide several benefits to developing infants, including the recruitment of beneficial bacteria to the human gut. Akkermansia strains are largely considered beneficial bacteria and have been detected in colostrum, breast milk, and young infants. A. muciniphila MucT, belonging to the AmI phylogroup, contributes to the HMO deconstruction capacity of the infant. Here, using phylogenomics, we examined the genomic capacities of four Akkermansia phylogroups to deconstruct HMOs. Indeed, each phylogroup contained differences in their genomic capacities to deconstruct HMOs, and representative strains of each phylogroup were able to grow using HMOs. These Akkermansia-HMO interactions potentially influence gut microbial ecology in early life, a critical time for the development of the gut microbiome and infant health.

Complete Genome Sequence of Enterobacter asburiae Strain AEB30, Determined Using Illumina and PacBio Sequencing.

The complete genome sequence of Enterobacter asburiae strain AEB30 is presented. The strain was isolated from store-bought ginger in Albany, CA, in 2016.

Complete Genome Sequence of Pantoea agglomerans ASB05 Using Illumina and PacBio Sequencing.

We present the complete genome sequence of Pantoea agglomerans ASB05 and three associated plasmids, generated using a combination of the Illumina and PacBio platforms. P. agglomerans ASB05 was isolated from fresh cherries purchased in Albany, CA, in 2016.

Genomic Characterization of Campylobacter jejuni Adapted to the Guinea Pig (Cavia porcellus) Host.

Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide with excessive incidence in low-and middle-income countries (LMIC). During a survey for C. jejuni from putative animal hosts in a town in the Peruvian Amazon, we were able to isolate and whole genome sequence two C. jejuni strains from domesticated guinea pigs (Cavia porcellus). The C. jejuni isolated from guinea pigs had a novel multilocus sequence type that shared some alleles with other C. jejuni collected from guinea pigs. Average nucleotide identity and phylogenetic analysis with a collection of C. jejuni subsp. jejuni and C. jejuni subsp. doylei suggest that the guinea pig isolates are distinct. Genomic comparisons demonstrated gene gain and loss that could be associated with guinea pig host specialization related to guinea pig diet, anatomy, and physiology including the deletion of genes involved with selenium metabolism, including genes encoding the selenocysteine insertion machinery and selenocysteine-containing proteins.

Genomic Characterization of Salmonella typhimurium DT104 Strains Associated with Cattle and Beef Products.

Salmonella enterica subsp. enterica serovar Typhimurium DT104, a multidrug-resistant phage type, has emerged globally as a major cause of foodborne outbreaks particularly associated with contaminated beef products. In this study, we sequenced three S. Typhimurium DT104 strains associated with a 2009 outbreak caused by ground beef, including the outbreak source strain and two clinical strains. The goal of the study was to gain a stronger understanding of the genomics and genomic epidemiology of highly clonal S. typhimurium DT104 strains associated with bovine sources. Our study found no single nucleotide polymorphisms (SNPs) between the ground beef source strain and the clinical isolates from the 2009 outbreak. SNP analysis including twelve other S. typhimurium strains from bovine and clinical sources, including both DT104 and non-DT104, determined DT104 strains averaged 55.0 SNPs between strains compared to 474.5 SNPs among non-DT104 strains. Phylogenetic analysis separated the DT104 strains from the non-DT104 strains, but strains did not cluster together based on source of isolation even within the DT104 phage type. Pangenome analysis of the strains confirmed previous studies showing that DT104 strains are missing the genes for the allantoin utilization pathway, but this study confirmed that the genes were part of a deletion event and not substituted or disrupted by the insertion of another genomic element. Additionally, cgMLST analysis revealed that DT104 strains with cattle as the source of isolation were quite diverse as a group and did not cluster together, even among strains from the same country. Expansion of the analysis to 775 S. typhimurium ST19 strains associated with cattle from North America revealed diversity between strains, not limited to just among DT104 strains, which suggests that the cattle environment is favorable for a diverse group of S. typhimurium strains and not just DT104 strains.

DNA adenine methylase, not the PstI restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coli.

We previously reported a distinct methylome between the two Shiga toxin-producing Escherichia coli (STEC) O145:H28 strains linked to the 2010 U.S. lettuce-associated outbreak (RM13514) and the 2007 Belgium ice cream-associated outbreak (RM13516), respectively. This difference was thought to be attributed to a prophage encoded type II restriction-modification system (PstI R-M) in RM13514. Here, we characterized this PstI R-M system in comparison to DNA adenine methylase (Dam), a highly conserved enzyme in γ proteobacteria, by functional genomics. Deficiency in Dam led to a differential expression of over 1000 genes in RM13514, whereas deficiency in PstI R-M only impacted a few genes transcriptionally. Dam regulated genes involved in diverse functions, whereas PstI R-M regulated genes mostly encoding transporters and adhesins. Dam regulated a large number of genes located on prophages, pathogenicity islands, and plasmids, including Shiga toxin genes, type III secretion system (TTSS) genes, and enterohemolysin genes. Production of Stx2 in dam mutant was significantly higher than in RM13514, supporting a role of Dam in maintaining lysogeny of Stx2-prophage. However, following mitomycin C treatment, Stx2 in RM13514 was significantly higher than that of dam or PstI R-M deletion mutant, implying that both Dam and PstI R-M contributed to maximum Stx2 production.

Effect of the electron donating group on the excited-state electronic nature and epsilon-near-zero properties of curcuminoid-borondifluoride dyes.

Epsilon-near-zero (ENZ) properties have been reported in organic molecular films. In particular, cyanine and squaraine films have been shown to exhibit ENZ properties in the visible spectral region with a strong 3rd order nonlinear optical response near the ENZ spectral region. Noting both cyanine and squaraine belong to the polymethine family, a series of six curcuminoid borondifluoride (Curc) derivatives were developed to examine whether such a polymethine character is positively correlated with the ENZ property of the organic films. Those Curc derivatives possess a Donor-Acceptor-Donor (D-A-D) architecture with acceptor, AcacBF2, located at the molecular center. The backbone of Curc is designed such that the donor strength can be tuned to transit between charge transfer (CT) and polymethine character. This balance between CT and polymethine character of the Curc series is examined based on the Lippert-Mataga plot. As donor strength in the D-A-D structure increases, CT character is less marked resulting in a more dominant polymethine character. The structural and optical properties of the Curc films with a thickness in the order of 30 nm were examined to correlate the polymethine character with the ENZ response. The results obtained in isotropic Curc thin films demonstrate that an increase of polymethine character associated with a stronger donor strength leads to an appearance/enhancement of the ENZ property in the visible spectrum range from 500 to 670 nm. Overall, this study provides useful guidelines to engineer new organic materials showing ENZ properties in a desired spectral range.

Dataset of the phospholipidome and transcriptome of Campylobacter jejuni under different growth conditions.

The membrane phospholipid composition is not a stable bacterial characteristic but can change in response to altered environmental conditions. Here we provide the dataset of the phospholipidome and transcriptome of the microaerophilic human pathogen Campylobacter jejuni under different environmental conditions. These data have been used in Cao (2020), The unique phospholipidome of the enteric pathogen C. jejuni: Lysolipids are required for motility at low oxygen availability. Here the abundance of each phospholipid is shown during the growth of C. jejuni for 0-108 h under low and high oxygen conditions (0.3 vs 10% O2). The phospholipid data were obtained by applying high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS). The transcriptomic data obtained by RNA-seq show the differential expressed genes between logarithmic and stationary grown bacteria. In addition, our data might serve as a reference information for further in-depth investigation to understand the relation between specific phospholipids and the activity of membrane associated proteins.

A porcine ligated loop model reveals new insight into the host immune response against Campylobacter jejuni.

The symptoms of infectious diarrheal disease are mediated by a combination of a pathogen's virulence factors and the host immune system. Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide due to its near-ubiquitous zoonotic association with poultry. One of the outstanding questions is to what extent the bacteria are responsible for the diarrheal symptoms via intestinal cell necrosis versus immune cell initiated tissue damage. To determine the stepwise process of inflammation that leads to diarrhea, we used a piglet ligated intestinal loop model to study the intestinal response to C. jejuni. Pigs were chosen due to the anatomical similarity between the porcine and the human intestine. We found that the abundance of neutrophil related proteins increased in the intestinal lumen during C. jejuni infection, including proteins related to neutrophil migration (neutrophil elastase and MMP9), actin reorganization (Arp2/3), and antimicrobial proteins (lipocalin-2, myeloperoxidase, S100A8, and S100A9). The appearance of neutrophil proteins also corresponded with increases of the inflammatory cytokines IL-8 and TNF-α. Compared to infection with the C. jejuni wild-type strain, infection with the noninvasive C. jejuni ∆ciaD mutant resulted in a blunted inflammatory response, with less inflammatory cytokines and neutrophil markers. These findings indicate that intestinal inflammation is driven by C. jejuni virulence and that neutrophils are the predominant cell type responding to C. jejuni infection. We propose that this model can be used as a platform to study the early immune events during infection with intestinal pathogens.

Unique inducible filamentous motility identified in pathogenic Bacillus cereus group species.

Active migration across semi-solid surfaces is important for bacterial success by facilitating colonization of unoccupied niches and is often associated with altered virulence and antibiotic resistance profiles. We isolated an atmospheric contaminant, subsequently identified as a new strain of Bacillus mobilis, which showed a unique, robust, rapid, and inducible filamentous surface motility. This flagella-independent migration was characterized by formation of elongated cells at the expanding edge and was induced when cells were inoculated onto lawns of metabolically inactive Campylobacter jejuni cells, autoclaved bacterial biomass, adsorbed milk, and adsorbed blood atop hard agar plates. Phosphatidylcholine (PC), bacterial membrane components, and sterile human fecal extracts were also sufficient to induce filamentous expansion. Screening of eight other Bacillus spp. showed that filamentous motility was conserved amongst B. cereus group species to varying degrees. RNA-Seq of elongated expanding cells collected from adsorbed milk and PC lawns versus control rod-shaped cells revealed dysregulation of genes involved in metabolism and membrane transport, sporulation, quorum sensing, antibiotic synthesis, and virulence (e.g., hblA/B/C/D and plcR). These findings characterize the robustness and ecological significance of filamentous surface motility in B. cereus group species and lay the foundation for understanding the biological role it may play during environment and host colonization.