Genomic analysis of a cAmpC (CMY-41)-producing Citrobacter freundii ST64 isolated from patient.

Antibiotic resistance in Citrobacter freundii is a public health concern. This study evaluated the closed genome of a C. freundii isolated from the stool of a hospitalized patient initially related to a Salmonella outbreak. Confirmation of the isolate was determined by whole-genome sequencing. Nanopore sequencing was performed using a MinION with a Flongle flow cell. Assembly using SPAdes and Unicycler yielded a closed genome annotated by National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline. Genomic analyses employed MLST 2.0, ResFinder4.1, PlasmidFinder2.1, and VFanalyzer. Phylogenetic comparison utilized the Center for Food Safety and Applied Nutrition (CFSAN)-single nucleotide polymorphism pipeline and Genetic Algorithm for Rapid Likelihood Inference. Antimicrobial susceptibility was tested by broth microdilution following Clinical and Laboratory Standards Institute criteria. Multi-locus sequence type in silico analysis assigned the C. freundii as sequence type 64 and the blaCMY-41 gene was detected in resistome investigation. The susceptibility to antibiotics, determined using Sensititre® plates, revealed resistance to aztreonam, colistin, cefoxitin, amoxicillin/clavulanic acid, sulfisoxazole, ampicillin, and streptomycin. The genetic relatedness of the C. freundii CFSAN077772 with publicly available C. freundii genomes revealed a close relationship to a C. freundii SRR1186659, isolated in 2009 from human stool in Tanzania. In addition, C. freundii CFSAN077772 is nested in the same cluster with C. freundii clinical strains isolated in Denmark, Mexico, Myanmar, and Canada, suggesting a successful intercontinental spread.

Longitudinal Survey of Aeromonas hydrophila and Foodborne Pathogens in a Commercial Aquaponics System.

Aquaponic production of fresh produce is a sustainable agricultural method becoming widely adopted, though few studies have investigated potential food safety hazards within commercial systems. A longitudinal study was conducted to isolate and quantify several foodborne pathogens from a commercial, aquaponic farm, and to elucidate their distribution throughout. The survey was conducted over 2 years on a controlled-environment farm containing Nile tilapia (Oreochromis niloticus) and lettuce (Lactuca sativa). Samples (N = 1,047) were collected bimonthly from three identical, independent systems, and included lettuce leaves, roots, fingerlings (7-126 d old), feces from mature fish (>126 d old), water, and sponge swabs collected from the tank interior surface. Most probable number of generic Escherichia coli were determined using IDEXX Colilert Quanti-Tray. Enumeration and enrichment were used to detect Shiga toxin-producing E. coli (STEC), Salmonella enterica, Listeria monocytogenes, Aeromonas spp., Aeromonas hydrophilia, and Pseudomonas aeruginosa. Generic E. coli, STEC, L. monocytogenes, and S. enterica were not detected in collected samples. P. aeruginosa was isolated from water (7/351; 1.99%), swabs (3/351; 0.85%), feces (2/108; 1.85%), and lettuce leaves (2/99; 2.02%). A. hydrophila was isolated from all sample types (623/1047; 59.50%). The incidence of A. hydrophila in water (X2 = 23.234, p < 0.001) and sponge samples (X2 = 21.352, p < 0.001) increased over time.

Effect of aquaponic water and substratum material on biofilm formation by Aeromonas hydrophila.

Aeromonas hydrophila is a zoonotic pathogen causing illness in fish and susceptible humans. This emerging pathogen has been isolated within aquaponic systems and could cause disease in fish and a hazard to humans consuming aquaponic produce. This study determined whether A. hydrophila from an aquaponic farm could form biofilms in aquaponic water and on materials used in these systems. A. hydrophila biofilm biomass and cell density in aquaponic water were evaluated by crystal violet staining and culture-based enumeration. Biofilm biomass and biofilm cell density were affected by the water source and A. hydrophila isolate (P < 0.05). A. hydrophila formed the most biomass from the beginning of deep-water culture (BDWC) water (OD570 0.202 ± 0.066) and the least from the end of deep-water culture (EDWC) water (OD570 0.140 ± 0.036; P < 0.05). Enumerated A. hydrophila from the biofilm varied among water sources; the fish tank water supported the greatest cell density (7.04 ± 0.71 log CFU/mL) while the EDWC supported the lowest cell density (6.76 ± 0.83 log CFU/mL). Biofilm formation was also evaluated on aquaponic materials such as nylon, polyvinyl chloride, polyethylene liner, bead filter, and foam. Biofilm formation on the liner had the greatest population (2.39 ± 0.022 log CFU/cm2), and the bead had the least (0.64 ± 0.039 log CFU/cm2; P < 0.05). Pathogenic organisms, such as A. hydrophila, may pose a greater risk to produce harvested from the BDWC and MDWC due to greater biofilm formation.

The composition of environmental microbiota in three tree fruit packing facilities changed over seasons and contained taxa indicative of L. monocytogenes contamination.

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.

qPCR detection of viable Bacillus cereus group cells in cosmetic products.

Reference methods for microbiological safety assessments of cosmetics rely on culture methods that reveal colonies of live microorganisms on growth media. Rapid molecular technologies, such as qPCR, detects the presence of target DNA in samples from dead and viable cells. DNA intercalating dyes, such as propidium monoazide (PMAxx), are capable of restricting PCR amplification to viable microbial cells. Here we developed singleplex and multiplex real time (qPCR) assays for the detection of Bacillus cereus (B. cereus) using 16S rRNA and phosphatidylcholine-specific phospholipase C (PLC) gene specific sequences coupled with PMAxx. The limit of detection was determined to be ~ 1 log CFU/ml for 16S rRNA and 3 log CFU/ml for PLC detection in pure culture using an eye shadow isolate, B. cereus 3A. We assessed the inclusivity and exclusivity of our qPCR assays using 212 strains, including 143 members of B. cereus, 38 non- B. cereus. and 31 non-Bacillus species; inclusivity was 100% for the 16S rRNA and 97.9% for the PLC targets; the exclusivity was 100% for 16S rRNA and 98.6% for PLC targets. These qPCR assays were then used to assess samples of commercial cosmetics: one set of liquid face toners (N = 3), artificially contaminated with B. cereus 3A, and one set of powdered cosmetics (N = 8), previously determined to be contaminated with B. cereus. For some samples, test portions were analyzed by qPCR in parallel, with and without PMAxx treatment. All test portions were simultaneously streaked on BACARA plates to confirm viable cells of B. cereus, according to the culture method. We found no difference in sensitivity between the singleplex and the multiplex qPCR assays (P > 0.05). Inoculated samples that did not recover B. cereus on plates still showed amplification of the DNA targets. However, that amplification was significantly delayed in PMAxx -treated samples (P < 0.0001) with CT value differences of 7.82 for 16S rRNA and 7.22 for PLC. Likewise, amplification delay was significant (P < 0.0001) with inoculated samples that recovered B. cereus on plates with CT value differences of 2.96 and 2.36 for 16S rRNA and PLC, respectively, demonstrating the presence of dead cells in the samples. All our qPCR results correlated with detection on BACARA plates (kappa, k = 0.99), independently of the presence of PMAxx in the PCR assays. Nevertheless, the amplification threshold with PMAxx dyes was significantly higher than the non-PMAxx dyes. Our findings confirm qPCR can be used for more rapid detection of microorganisms in cosmetics, including B. cereus, and selective detection of viable cells can be improved using PMAxx dyes.

Genomic and phylogenetic analysis of Salmonella enterica serovar Enteritidis strains linked to multiple outbreaks in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis (SE) has become the prevalent serovar isolated from gastroenteritis cases in Brazil since the 1990s. To better understand the genomic diversity and phylogenetic relationship amongst SE epidemic isolates from Brazil, 30 SE isolates from a variety of implicated foods and case patients of outbreaks between 1999 and 2006 were selected for genome comparison analyses. SE genomes were also compared against publicly available Brazilian SE isolates from pre- and postepidemic period. MLST analysis revealed that all isolates belong to sequence type (ST) 11. A total of seven Salmonella pathogenicity islands (SPIs) (SPI-1, SPI-3-5, SPI-13, SPI14, and C63PI) were identified in the evaluated genomes and all studied SE genomes carried similar prophage profiling. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac(6')laa, aph(3")-lb, aph(6)-ld], as well as point mutations in gyrA. Phylogenetic analysis demonstrated that certain isolates have circulated in Brazil for years and been involved in distinct outbreaks.

Exploring the antimicrobial effects of a phenolic-rich extract from jabuticaba depulping waste against enterotoxigenic Escherichia coli.

This study evaluated the effects of a phenolic-rich extract from jabuticaba [Myrciaria jaboticaba (Vell.) Berg] depulping waste (PEJ) on the survival, antibiotic susceptibility, virulence, and cellular functions of various enterotoxigenic Escherichia coli (ETEC) strains. The minimum inhibitory concentration of PEJ against the five tested ETEC strains was 125 mg mL-1. PEJ at 125 and 250 mg mL-1 caused reductions in viable cell counts of ≥ 3 and ≥ 5 log CFU mL-1 in ETEC over 24 h, respectively. PEJ at subinhibitory concentrations (31.25 and 62.5 mg mL-1) reduced the viable cell counts of ETEC when exposed to in vitro gastrointestinal conditions, besides decreasing the biofilm formation, cell surface hydrophobicity, mucin adhesion, and swimming and swarming motility. PEJ (31.25 and 62.5 mg mL-1) increased the susceptibility of the tested ETEC strains to various clinically relevant antibiotics. The exposure to PEJ (62.5 and 125 mg mL-1) impaired the membrane permeability and enzymatic and efflux pump activities in ETEC cells. PEJ effectively reduces survival, increases antibiotic susceptibility, and attenuates virulence in ETEC. These effects could be linked to a PEJ multi-target action disturbing various cellular functions in ETEC cells. PEJ could be a candidate for developing innovative solutions to prevent and treat ETEC infections.

The Persistence of Bacterial Pathogens in Surface Water and Its Impact on Global Food Safety.

Water is vital to agriculture. It is essential that the water used for the production of fresh produce commodities be safe. Microbial pathogens are able to survive for extended periods of time in water. It is critical to understand their biology and ecology in this ecosystem in order to develop better mitigation strategies for farmers who grow these food crops. In this review the prevalence, persistence and ecology of four major foodborne pathogens, Shiga toxin-producing Escherichia coli (STEC), Salmonella, Campylobacter and closely related Arcobacter, and Listeria monocytogenes, in water are discussed. These pathogens have been linked to fresh produce outbreaks, some with devastating consequences, where, in a few cases, the contamination event has been traced to water used for crop production or post-harvest activities. In addition, antimicrobial resistance, methods improvements, including the role of genomics in aiding in the understanding of these pathogens, are discussed. Finally, global initiatives to improve our knowledge base of these pathogens around the world are touched upon.

Prevalence and Distribution of Listeria monocytogenes in Three Commercial Tree Fruit Packinghouses.

A 2-year longitudinal study of three tree fruit packinghouses was conducted to determine the prevalence and distribution of Listeria monocytogenes. Samples were collected from 40 standardized non-food-contact surface locations six different times over two 11-month production seasons. Of the 1,437 samples collected, the overall prevalence of L. monocytogenes over the course of the study was 17.5%. Overall prevalence did not differ significantly (p > 0.05) between each year. However, values varied significantly (p ≤ 0.05) within each production season following packing activity levels; increasing in the fall, peaking in early winter, and then decreasing through spring. L. monocytogenes was most often found in the packing line areas, where moisture and fruit debris were commonly observed and less often in dry cold storage and packaging areas. Persistent contamination was attributed to the inability of water drainage systems to prevent moisture accumulation on floors and equipment during peak production times and uncontrolled employee and equipment traffic throughout the facility. This is the first multiyear longitudinal surveillance study to compare L. monocytogenes prevalence at standardized sample sites common to multiple tree fruit packinghouses. Recommendations based on our results will help packinghouse operators to identify critical areas for inclusion in their L. monocytogenes environmental monitoring programs.

Genomic investigation of antimicrobial resistance determinants and virulence factors in Salmonella enterica serovars isolated from contaminated food and human stool samples in Brazil.

This study investigated the antimicrobial resistance determinants, virulence factors and identified serovars in 37 Salmonella enterica strains isolated from human stool and contaminated foods linked to outbreaks that occurred in Brazil over 7 years using whole genome sequencing (WGS). Phylogenetic analysis of selected serovars (S. Typhimurium, S. Infantis, S. London, and S. Johannesburg) was performed. Ten distinct serovars were identified and, 51% of the tested strains (n = 19) showed disagreement with the previous conventional serotyping. The antimicrobial resistance (AMR) determinants or plasmids varied among the strains. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac (6')-laa, aph (3″)-lb, aph (6)-ld, aadA1 and aadA2], sulfonamides (sul1), trimethoprin (dfrA8), fosfomycin (fosA7) and tetracyclines (tetA, tetB, tetC), as well as point mutations in parC (T57S) and gyrA (S83F). Plasmidome showed the presence of IncHI2, IncHI2A, IncFIB (S), IncFII (S), IncI1 and p0111 plasmids. Eight Salmonella pathogenicity islands and up to 102 stress and/or virulence genes were identified in the evaluated genomes. Virulence genes of K88 fimbrial adhesin were first reported in S. enterica (S. Pomona, S. Bredeney and S. Mbandaka strains). pilW gene was first identified in S. Pomona. Phylogenetic analysis showed that some serovars circulated in Brazil for decades, primarily within the poultry production chain. Findings highlighted the virulence and AMR determinants in strains that may lead to recurring food outbreaks.

Genetic Diversity of Listeria monocytogenes Isolated From Three Commercial Tree Fruit Packinghouses and Evidence of Persistent and Transient Contamination.

Whole genome analysis was performed on 501 isolates obtained from a previous survey which recovered 139 positive environmental sponge samples (i.e., up to 4 isolates per sample) from a total of 719 samples collected at 40 standardized sites in 3 commercial apple packinghouse facilities (i.e., P1, P2, and P3) over 3 successive seasons in a single production year. After excluding duplicated isolates, the data from 156 isolates revealed the clonal diversity of L. monocytogenes and allowed the detection of transient contamination, persistent contamination, and cross-area transmission events. Facility P2 with the poorest sanitary conditions had the least diversity (Shannon's index of 0.38). P2 contained a Clonal Complex (CC) 554, serogroup IVb-v1 strain that persisted throughout the year and spread across the entire facility, a singleton Sequence Type (ST) 1003, lineage III strain that persisted through two seasons and spread across two areas of the facility, and 3 other clones from transient contaminations. P1 and P3, facilities with better sanitary conditions, had much higher diversity (i.e., 15 clones with a Shannon's index of 2.49 and 10 clones with a Shannon's index of 2.10, respectively) that were the result of transient contamination. Facilities P1 and P3 had the highest incidence (43.1%) of lineage III isolates, followed by lineage I (31.3%) and lineage II (25.5%) isolates. Only 1 isolate in the three facilities contained a premature stop codon in virulence gene inlA. Fourteen samples yielded 2-3 clones per sample, demonstrating the importance of choosing appropriate methodologies and selecting a sufficient number of isolates per sample for studying L. monocytogenes diversity. Only 1 isolate, belonging to CC5 and from facility P3, contained a known plasmid, and this was also the only isolate containing benzalkonium chloride tolerance genes. The persistent CC554 strain did not exhibit stronger sanitizer resistance than other isolates and did not contain any confirmed molecular determinants of L. monocytogenes stress resistance that were differentially present in other isolates, such as genes involved in sanitizer tolerance, heavy metal resistance, biofilm-forming, stress survival islet 1 (SSI-1), stress survival islet 2 (SSI-2) or Listeria genomic island (LGI2).

Survival of a serotype 4b strain and a serotype 1/2a strain of Listeria monocytogenes, isolated from a stone fruit outbreak investigation, on whole stone fruit at 4 °C.

In the summer of 2014, a multistate outbreak of listeriosis associated with contaminated stone fruit (peach and nectarine) was reported. A serotype 4b variant Listeria monocytogenes (Lm) strain of singleton Sequence Type (ST) 382 was isolated from clinical samples and stone fruit associated with the outbreak. A serotype 1/2b Lm strain of ST5, Clonal Complex 5 was isolated only from outbreak-associated stone fruit, not from clinical samples. Here we investigated the fate of the serotype 4b and 1/2b strains, at two inoculation levels (high level at 3.7 logCFU/fruit and low level at 2.7 logCFU/fruit), on the surfaces of white peach, yellow peach and yellow nectarine stored at 4 °C for 26 days. After rinsing the fruits, we determined the Lm levels in the rinsates and on the peels. We enumerated Lm using a direct plating method and compared two chromogenic agars. The Lm populations rapidly declined in the first 3 days and then declined more slowly until Day 19/21. The maximum decline was 1.6 logCFU/fruit on yellow peach inoculated with serotype 4b at high level. For fruits inoculated with high-level Lm, the lowest level of Lm (1.7 logCFU/fruit) was observed on for white peach inoculated with serotype 1/2b, and the highest level of Lm (2.6 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with the serotype 1/2b strain. For fruits inoculated with low-level Lm, the lowest level of Lm (1.3 logCFU/fruit) was observed on yellow nectarine inoculated with either the serotype 4b or 1/2b strain, and the highest level of Lm (1.7 logCFU/fruit) on Day 19/21 was observed on yellow peach inoculated with ST382. The D-values ranged from 15 days to 28 days. Lm remained viable until the end of storage (Day 26), but the levels were not significantly different from those on Day 19/21. The types of stone fruit and Lm strain did not significantly affect the survival of Lm. These results demonstrate that contaminated stone fruit can carry a potential risk for causing listeriosis in susceptible populations. Comparison of direct plating results using two chromogenic agars showed that RAPID' L. mono and Agar Listeria Ottavani & Agosti performed equivalently for enumerating Lm on stone fruit. The fruit rinsing recovered 80% to 84% of Lm from fruit surfaces.

Antimicrobial Efficacy of Pelargonic Acid Micelles against Salmonella varies by Surfactant, Serotype and Stress Response.

The antimicrobial properties of Pelargonic acid (PA), a component of tomatoes, makes it an attractive candidate as a food additive and sanitizer. The antimicrobial efficacy of PA emulsions generated using surfactants: Tween 80, Triton X100, Sodium Dodecyl Sulfate (SDS) and Quillaja Saponin was evaluated against Salmonella serotypes Newport, Oranienburg and Typhimurium. Micelle/dropletsize, and minimal inhibitory concentrations (MIC) were determined. Surfactant type and concentration significantly influenced the antimicrobial efficacy of PA (p < 0.05). Overall, Salmonella Newport was the most (p < 0.05) susceptible serotype to PA emulsions. PA emulsions generated with 1.00% SDS had the highest (p < 0.05) antimicrobial activity, with MIC of 7.82 mM against S. Newport and 15.62 mM against S. Oranienburg/S. Typhimurium, respectively. Addition of PA to Trypticase Soy Broth resulted in a decreased growth rate and an increased lag phase duration. Cells exposed to PA formed elongated filaments (>5 µm). Additionally, Salmonella serotypes Typhimurium and Newport also formed floccular biofilms. PA emulsions at a concentration of 31.25 mM generated using 1% SDS and 1% Quillaja saponin resulted in >6 log CFU/ml reduction in Salmonella population. Althought all PA emulsions evalauted inhibited Salmonella, morphological changes to this antimicrobial varied substantially among the Salmonella serotypes tested.

Effect of Washing, Waxing and Low-Temperature Storage on the Postharvest Microbiome of Apple.

There is growing recognition of the role that the microbiome plays in the health and physiology of many plant species. However, considerably less research has been conducted on the postharvest microbiome of produce and the impact that postharvest processing may have on its composition. Here, amplicon sequencing was used to study the effect of washing, waxing, and low-temperature storage at 2 °C for six months on the bacterial and fungal communities of apple calyx-end, stem-end, and peel tissues. The results of the present work reveal that tissue-type is the main factor defining fungal and bacterial diversity and community composition on apple fruit. Both postharvest treatments and low temperature storage had a strong impact on the fungal and bacterial diversity and community composition of these tissue types. Distinct spatial and temporal changes in the composition and diversity of the microbiota were observed in response to various postharvest management practices. The greatest impact was attributed to sanitation practices with major differences among unwashed, washed and washed-waxed apples. The magnitude of the differences, however, was tissue-specific, with the greatest impact occurring on peel tissues. Temporally, the largest shift occurred during the first two months of low-temperature storage, although fungi were more affected by storage time than bacteria. In general, fungi and bacteria were impacted equally by sanitation practices, especially the epiphytic microflora of peel tissues. This research provides a foundation for understanding the impact of postharvest management practices on the microbiome of apple and its potential subsequent effects on postharvest disease management and food safety.

Successive exposure to Mentha piperita L. essential oil affects the culturability and induces membrane repair in a persister epidemic Salmonella Typhimurium PT4.

This study had as aims to evaluate the effects of successive exposures to Mentha piperita L. essential oil (MPEO) on culturability and physiological functions of Salmonella Typhimurium PT4. S. Typhimurium PT4 cells (108 log CFU/mL) were exposed to the same (1.25 µL/mL) or increasing MPEO concentrations (1.25-80 µL/mL) during 252 h. At each 36-h interval, the viable cell counts, and distinct cell functions were assessed using plate counting and flow cytometry, respectively. As the exposure time to the same MPEO concentration increased, the population of S. Typhimurium PT4 cells with damaged, permeabilized and depolarized membrane, and compromised efflux activity decreased. Otherwise, S. Typhimurium PT4 cells with damaged membrane physiological functions increased over the exposure to increasing concentrations of MPEO. Genomic analyses showed that the strain carries 17 genes associated with stress responses and the persistence of the tested strain among sources associated with poultry spanning more than 16 years and its virulence for humans. Therefore, successive exposure to a sublethal concentration of MPEO induced S. Typhimurium PT4 cells capable of maintaining the membrane integrity and its functions despite their non-culturable state.

Draft Genome Sequences of 26 Salmonella Isolates Associated with Multiple Outbreaks in Brazil.

Salmonella enterica subsp. enterica isolates are the leading cause of foodborne illness worldwide. Here, we report the draft genomes of 26 Salmonella isolates of food and clinical origin, belonging to four serovars, associated with outbreaks from 1999 to 2006 in the south of Brazil.

Draft Genome Sequences of 30 Salmonella enterica Serovar Enteritidis Isolates Associated with Multiple Outbreaks in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis has been the prevalent serovar isolated from gastroenteritis cases in Brazil since the 1990s. Here, we report the draft genomes of 30 S Enteritidis isolates originating from a variety of patients and implicated foods during outbreaks between 1999 and 2006 in Brazil.

Survival of outbreak, food, and environmental strains of Listeria monocytogenes on whole apples as affected by cultivar and wax coating.

The 2014-2015 U.S. nationwide outbreak of listeriosis linked to apples used in commercially produced, prepackaged caramel apples was the first implication of whole apples in outbreaks of foodborne illnesses. Two case patients of this outbreak didn't consume caramel apples but did eat whole apples, suggesting that contaminated whole apple may serve as a vehicle for foodborne listeriosis. The current study evaluated the effect of conventional fruit coating with wax and that of apple cultivar on the survival of outbreak-associated and non-outbreak Listeria monocytogenes strains on Red Delicious, Granny Smith and Fuji apples during 160 days under simulated commercial storage. L. monocytogenes survived in calyxes and stem ends of apples of all 3 cultivars through the duration of the experiment. After 2 months of storage, significantly (p < 0.05) larger L. monocytogenes populations were recovered from apples coated with wax than those un-waxed, regardless of the cultivar. No differences in survival amongst L. monocytogenes strains (serotypes 1/2a and 4b) from clinical, food, and environmental sources were observed. The observation that coating with wax facilitates prolonged survival of L. monocytogenes on whole apples is novel and reveals gaps in understanding of microbiological risks associated with postharvest practices of tree fruit production.

The occurrence of Listeria monocytogenes is associated with built environment microbiota in three tree fruit processing facilities.

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.

Salmonella enterica Filamentation Induced by Pelargonic Acid Is a Transient Morphotype.

Under stressful conditions, Salmonella enterica forms multinucleated elongated filaments. The triggers and outcomes of filamentation are not well characterized. S. enterica serotypes Newport, Javiana, and Typhimurium were evaluated for their ability to form filaments upon exposure to 20 mM pelargonic acid. S. Newport was used as a model to investigate the progression and fate of filamentation via culturable population size, cell length, and viability assays. All serotypes displayed filament formation after 16 h of incubation. Pelargonic acid amendment of tryptic soy broth (TSBpel) produced a 5-log CFU reduction compared to TSB after 24 h (P < 0.05), and the growth rate decreased (P < 0.02). Cell elongation started within 12 h, peaked at 16 h, and was followed by filament disintegration at 20 to 24 h. The ratio of filaments to regular-sized cells (F/R) in TSBpel was 3.87 ± 0.59 at 16 h, decreasing to 0.23 ± 0.04 and 0.03 ± 0.01 (P < 0.05) at 20 and 24 h, respectively. Mg2+ supplementation repressed filamentation (F/R = 0.25 ± 0.11) and enhanced culturable cell counts (P < 0.05). Continued exposure to pelargonic acid inhibited growth in TSB and M9 compared to that in unamended media (P < 0.05). However, in M9 medium without Mg2+ amended with 20 mM pelargonic acid (M9pel), filament fragmentation progressed independently of pelargonic acid or Mg2+ When cells were pretreated with pelargonic acid to induce filamentation and then transferred to fresh medium, a positive effect of Mg2+ was noted under nutrient-deficient conditions, with higher live/dead cell ratios in M9 supplemented with 5 mM Mg2+ (M9Mg) than in M9 (P < 0.05). No change was observed when pelargonic acid was also added. Filamentation was ubiquitous in all serotypes tested, transient, and sensitive to Mg2+ Fragmentation, but not recovery, progressed irrespective of antimicrobial or Mg2+ presence.IMPORTANCE Some bacteria form elongated multinucleated structures, or filaments, when exposed to stress. The filamentous form of foodborne bacterial pathogens can interfere with food protection practices and diagnostic testing. Filamentation in Salmonella enterica Newport was investigated in response to pelargonic acid, a compound naturally found in several fruit and vegetables, and also used commercially as an herbicide. Salmonella readily formed filaments when exposed to pelargonic acid. Filaments were not stable, however, and fragmented to individual cells even when the fatty acid was still present, recovering fully when the stress was alleviated. A deeper exploration of the molecular mechanisms regulating filamentation and the conditions that induce it in agriculture and the food supply chain is needed to devise strategies that curb this response.