Enrichment dynamics of Listeria monocytogenes and the associated microbiome from naturally contaminated ice cream linked to a listeriosis outbreak.

BACKGROUND: Microbiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery. Here, dynamics of co-enriching microbiota during recovery of Listeria monocytogenes from naturally contaminated ice cream samples linked to an outbreak are described for three different initial enrichment formulations used by the Food and Drug Administration (FDA), the International Organization of Standardization (ISO), and the United States Department of Agriculture (USDA). Enrichment cultures were analyzed using DNA extraction and sequencing from samples taken every 4 h throughout 48 h of enrichment. Resphera Insight and CosmosID analysis tools were employed for high-resolution profiling of 16S rRNA amplicons and whole genome shotgun data, respectively. RESULTS: During enrichment, other bacterial taxa were identified, including Anoxybacillus, Geobacillus, Serratia, Pseudomonas, Erwinia, and Streptococcus spp. Surprisingly, incidence of L. monocytogenes was proportionally greater at hour 0 than when tested 4, 8, and 12 h later with all three enrichment schemes. The corresponding increase in Anoxybacillus and Geobacillus spp.indicated these taxa co-enriched in competition with L. monocytogenes during early enrichment hours. L. monocytogenes became dominant after 24 h in all three enrichments. DNA sequences obtained from shotgun metagenomic data of Listeria monocytogenes at 48 h were assembled to produce a consensus draft genome which appeared to have a similar tracking utility to pure culture isolates of L. monocytogenes. CONCLUSIONS: All three methods performed equally well for enrichment of Listeria monocytogenes. The observation of potential competitive exclusion of L. mono by Anoxybacillus and Geobacillus in early enrichment hours provided novel information that may be used to further optimize enrichment formulations. Application of Resphera Insight for high-resolution analysis of 16S amplicon sequences accurately identified L. monocytogenes. Both shotgun and 16S rRNA data supported the presence of three slightly variable genomes of L. monocytogenes. Moreover, the draft assembly of a consensus genome of L. monocytogenes from shotgun metagenomic data demonstrated the potential utility of this approach to expedite trace-back of outbreak-associated strains, although further validation will be needed to confirm this utility.

Evaluation of corn oil as an additive in the pre-enrichment step to increase recovery of Salmonella enterica from oregano.

Phenolic compounds associated with essential oils of spices and herbs possess a variety of antioxidant and antimicrobial properties that interfere with Salmonella detection from fresh and dried products. Finding a compound to neutralize the effect of these antimicrobial compounds, while allowing Salmonella growth during pre-enrichment, is a crucial step in both traditional pathogen isolation and molecular detection from these foods. This study evaluated the effectiveness of corn oil as a component of the pre-enrichment broth to counteract antimicrobial compounds properties and increase the recovery of Salmonella from spices. Oregano samples artificially contaminated with Salmonella enterica were pre-enriched in modified Buffered Peptone Water (mBPW) supplemented with and without 2% (vol/vol) corn oil respectively. Samples were incubated overnight at 37 °C. The results showed that recovery of Salmonella from oregano samples was increased by ≥50% when pre-enriched with corn oil. Serovars were confirmed using a PCR serotyping method. In addition, shot-gun metagenomics analyses demonstrated bacterial diversity and the effect of corn oil on the relative prevalence of Salmonella in the oregano samples. Modifying pre-enrichment broths with corn oil improved the detection and isolation of Salmonella from oregano, and may provide an alternative method for pathogen detection in dried food matrices such as spices.

Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing.

BACKGROUND: Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective pre-enrichment culture step commonly used by laboratory analysts to resuscitate microorganisms in foods suspected of contamination with pathogens. Cilantro samples were processed for Salmonella detection according to the method in the United States Food and Drug Administration Bacteriological Analytical Manual. Genomic DNA was extracted from culture supernatants prior to and after a 24-hour nonselective pre-enrichment step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen the libraries for Salmonella, as some samples were known to be culture positive. Additionally, culture positive cilantro samples were examined for the presence of Salmonella using shotgun metagenomics on the Illumina MiSeq. RESULTS: Time zero uncultured samples had an abundance of Proteobacteria while the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes. Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples revealed variable degrees of Salmonella contamination among the sequenced samples. CONCLUSIONS: Our cilantro study demonstrates the use of high-throughput sequencing to reveal the microbiome of cilantro, and how the microbiome changes during the culture-based protocols employed by food safety laboratories to detect foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to a predominance of Firmicutes suggests that changing our culture-based methods will improve detection sensitivity for foodborne enteric pathogens.

Comparison of Salmonella enterica serovar Bovismorbificans 2011 hummus outbreak strains with non-outbreak strains.

Eleven Salmonella enterica serovar Bovismorbificans isolates obtained from the U.S. District of Columbia during a 2011 hummus-associated foodborne outbreak were compared to 12 non-outbreak isolates. All isolates from the outbreak demonstrated a single PFGE pattern that was distinctly different from other isolates of S. Bovismorbificans as recorded in the PulseNet Database. Results from molecular analyses of the hummus-associated S. Bovismorbificans isolates indicate that the isolates from the outbreak were unique and have acquired an 80-90 kb plasmid. The impact of this study is that the information gained will add and expand our knowledge of diversity of the S. Bovismorbificans serovar.

Distribution and pharmacokinetics of double-radiolabeled endotoxin in the rat brain and peripheral organs.

The endotoxin, lipopolysaccharide (LPS), of Salmonella typhimurium was biosynthetically labeled with (3)H and (14)C incorporated into the fatty acyl chains and glucosamine residues, respectively. The radio-labeled LPS was isolated from the bacteria and then injected into Sprague-Dawley rats. The distribution of (14)C and (3)H-LPS in plasma and other organs was determined following intraperitoneal (IP) doses of (14)C and (3)H-LPS (200 µg/kg). Plasma concentrations of both fatty acyl chains and glucosamine residues were biphasic, with a relatively rapid decay followed by a slow decline for 48 h. Similar biphasic results were found in the peripheral organs (kidney and heart) and brain barrier tissues (meninges and choroid plexus). In other brain tissues (brain stem, caudate nucleus, hypothalamus, frontal cortex, cerebellum and hippocampus), the glucosamine residue was biphasic, whereas the fatty acyl chains showed accumulation. Highest concentrations of LPS were found in the plasma, spleen and the liver. In addition, in the liver, sustained elevations of (14)C-glucosamine and (3)H-fatty acyl chains were observed. This indicates LPS accumulation in the liver. By contrast, the spleen showed biphasic decay of glucosamine residues and accumulation of fatty acyl chains. In the brain barrier tissues, peak LPS concentrations were significantly reduced (about 70%) and were further reduced (about 95%) in other brain tissues. The high elevation of LPS in the spleen is considered indicative of an immune response. Our findings highlight the potential significant role of lipid A as shown with the sustained elevation of (3)H-fatty acyl chains in the brain.

Occurrence and antibiotic resistance of multiple Salmonella serotypes recovered from water, sediment and soil on mid-Atlantic tomato farms.

Salmonella outbreaks associated with the consumption of raw tomatoes have been prevalent in recent years. However, sources of Salmonella contamination of tomatoes remain poorly understood. The objectives of this study were to identify ecological reservoirs of Salmonella on tomato farms, and to test antimicrobial susceptibilities of recovered Salmonella isolates. Fourteen Mid-Atlantic tomato farms in the U.S. were sampled in 2009 and 2010. Groundwater, irrigation pond water, pond sediment, irrigation ditch water, rhizosphere and irrigation ditch soil, leaves, tomatoes, and swabs of harvest bins and worker sanitary facilities were analyzed for Salmonella using standard culture methods and/or a flow-through immunocapture method. All presumptive Salmonella isolates (n=63) were confirmed using PCR and the Vitek(®) 2 Compact System, and serotyped using the Premi(®)Test Salmonella and a conventional serotyping method. Antimicrobial susceptibility testing was carried out using the Sensititre™ microbroth dilution system. Four of the 14 farms (29%) and 12 out of 1,091 samples (1.1%) were found to harbor Salmonella enterica subsp. enterica. Salmonella was isolated by the immunocapture method from soil, while the culture method recovered isolates from irrigation pond water and sediment, and irrigation ditch water. No Salmonella was detected on leaves or tomatoes. Multiple serotypes were identified from soil and water, four of which-S. Braenderup, S. Javiana, S. Newport and S. Typhimurium-have been previously implicated in Salmonella outbreaks associated with tomato consumption. Resistance to sulfisoxazole was prevalent and some resistance to ampicillin, cefoxitin, amoxicillin/clavulanic acid, and tetracycline was also observed. This study implicates irrigation water and soil as possible reservoirs of Salmonella on tomato farms and irrigation ditches as ephemeral habitats for Salmonella. The findings point to the potential for pre-harvest contamination of tomatoes from contaminated irrigation water or from soil or water splash from irrigation ditches onto low-lying portions of tomato plants.

The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices.

The most commonly used method for serotyping Salmonella spp. is based on the Kaufmann-White scheme, and is composed of serological reactions using antibodies to LPS agglutinins. The multiplex PCR used in this investigation was established by Kim et al. to serotype the 30 most common clinical Salmonella serotypes, as determined by CDC. The PCR assay consists of two five-plex reactions and a single two-plex PCR reaction, based on six genetic loci from Salmonella enterica serotype Typhimurium and four loci from S. enterica serotype Typhi. In this investigation, we further evaluated the method for serotyping Salmonella spp. using a reference collection, environmental samples collected from a Mid-Atlantic region tomato farm study, four food matrices spiked with different Salmonella serotypes and a proficiency test. The PCR assay was first evaluated using DNA isolated from pure cultures of isolates obtained from various clinical and environmental samples, and then DNA isolated from broth cultures of food matrices of "Red round" and Roma tomatoes, Romaine lettuce, green onions and Serrano peppers spiked with serotypes Newport, Typhimurium, Javiana and Saintpaul, respectively. The results showed that the PCR assay correctly serotyped Salmonella spp. from the clinical, environmental, spiked food matrices, and proficiency test samples. These findings are significant because the PCR assay was successful in the identification of Salmonella in the spiked samples in a broth culture containing other non-salmonella organism. This method may be a useful resource for the food safety community.

Evaluation of the Impact of Varied Carvacrol Concentrations on Salmonella Recovery in Oregano and How Corn Oil Can Minimize the Effect of Carvacrol during Preenrichment.

Phenolic compounds, like carvacrol, in oregano interfere with the detection of foodborne pathogens such as Salmonella enterica. Carvacrol concentration varies based on plant cultivars and growth region. Six oregano cultivars were used to compare the impact of carvacrol concentration on Salmonella and to evaluate the effectiveness of corn oil to help increase Salmonella survival for detection. The results of Agilent 1200 series high-performance liquid chromatography analysis showed that carvacrol concentration in the six oregano cultivars ranged from 64 to 11,200 ppm. Oregano samples were artificially contaminated with S. enterica and were preenriched in Trypticase soy broth with or without 2% (v/v) corn oil. After 18 to 24 h at 37°C, aliquots were transferred to selective enrichment broths. Salmonella was recovered onto xylose lysine Tergitol 4 agar. Six Salmonella serovars were compared, and recovery varied based on carvacrol concentration and serovar. Samples with higher concentrations of carvacrol showed Salmonella recovery only when they were preenriched with corn oil. Based on metagenomic analysis, the microflora associated with the oregano also varied per cultivar. The results show that, as carvacrol levels increased, Salmonella survival decreased. However, the addition of corn oil to the preenrichment broth can minimize the antimicrobial effects of the phenolic compounds, thus allowing for increased detection of Salmonella from oregano cultivars.

Evaluation of a multiplex PCR method to serotype Salmonella in animal feeds pre-enrichment broth cultures.

The identification of Salmonella enterica serotypes remains a highly important public health concern for microbiological analysis of foods, feeds, and clinical samples. Outbreaks of human salmonellosis are sometimes linked to contact with infected animals and animal feeds. To possibly reduce the number of outbreaks, it is important to rapidly, efficiently detect Salmonella enterica in animal feeds and food products. A multiplex PCR for molecular serotyping of Salmonella enterica previously used in a single lab validation study for serotyping in multiple human food matrices was used in this investigation to evaluate the effectiveness of the multiplex PCR assay as serotyping method and screening tool for Salmonella in animal feeds. This approach is unique in that: •The multiplex PCR serotyping assay may be used for rapid screening and serotyping of Salmonella enterica from contaminated animal feed at the non-selective pre-enrichment step.•The assay may provide the serotype or identification of Salmonella in positive samples at concentration as low as 10 CFU/25 g after a 24 h non-selective pre-enrichment step.•In addition to the ability to serotype, this assay contains invA as an internal control for Salmonella positive identification. The invA shows positive indication for Salmonella outside of the 30 serotypic banding patterns.

Assessing the effect of oral exposure to Paenibacillus alvei, a potential biocontrol agent, in male, non-pregnant, pregnant animals and the developing rat fetus.

Paenibacillus alvei, a naturally occurring soil microorganism, may be used in the control and/or elimination of human/animal pathogens present on/within produce commodities associated with human consumption. The safety of oral exposure to P. alvei in male, nulliparous females, the pregnant dam and developing fetus was assessed. Adult male and female rats received a single oral dose (gavage) of P. alvei and tissues were collected at post exposure days 0, 3 and 14. To evaluate the effect of the test organism on fetal development, sperm positive female rats received the test organism every 3 days thereafter throughout gestation. As human exposure would be no more than 1 × 103 CFU/ml the following dose levels were evaluated in both study phases: 0 CFU/ml tryptic soy broth (negative control); 1 × 108 CFU/ml; 1 × 104 CFU/ml or 1 × 102 CFU/ml. Neither sex specific dose-related toxic effects (feed or fluid consumption, body weight gain, and histopathology) nor developmental/reproductive effects including the number of implantations, fetal viability, fetal weight, fetal length and effects on ossification centers were observed. The test organism did not cross the placenta and was not found in the amniotic fluid.

Detection of Salmonella enterica subsp. enterica Serovar Cubana from Naturally Contaminated Chick Feed.

Because some significant outbreaks of human salmonellosis have been traced to contaminated animal feed, the rapid and efficient detection of Salmonella in feed is essential. However, the current U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) culture method that uses lactose broth as a preenrichment medium has not reliably supported the results of real-time PCR assays for certain foods. We evaluated the BAM culture method and a quantitative real-time PCR (qPCR) assay using two preenrichment media, modified buffered peptone water and lactose broth, to detect Salmonella enterica subsp. enterica serovar Cubana in naturally contaminated chick feed. After 24 h of incubation, the qPCR method was as sensitive as the culture method when modified buffered peptone water was used as the preenrichment medium but less sensitive than culture when lactose broth was used. After 48 h of incubation, detection of Salmonella Cubana by qPCR and by culture in either preenrichment medium was equivalent. We also compared the performance of the traditional serotyping method, which uses pure cultures of Salmonella grown on blood agar, to two molecular serotyping methods. The serotyping method based on whole genome sequencing also requires pure cultures, but the PCR-based molecular serotyping method can be done directly with the enriched culture medium. The PCR-based molecular serotyping method provided simple and rapid detection and identification of Salmonella Cubana. However, whole genome sequencing allows accurate identification of many Salmonella serotypes and highlights variations in the genomes, even in tight genomic clusters. We also compared the genome of the chick feed isolate with 58 Salmonella Cubana strains in GenBank and found that the chick feed isolate was very closely related to an isolate from a foodborne outbreak involving alfalfa sprouts.

Early Recovery of Salmonella from Food Using a 6-Hour Non-selective Pre-enrichment and Reformulation of Tetrathionate Broth.

Culture based methods are commonly employed to detect pathogens in food and environmental samples. These methods are time consuming and complex, requiring multiple non-selective and selective enrichment broths, and usually take at least 1 week to recover and identify pathogens. Improving pathogen detection in foods is a primary goal for regulatory agencies and industry. Salmonella detection in food relies on a series of culture steps in broth formulations optimized to resuscitate Salmonella and reduce the abundance of competitive bacteria. Examples of non-selective pre-enrichment broths used to isolate Salmonella from food include Lactose, Universal Pre-enrichment, BPW, and Trypticase Soy broths. Tetrathionate (TT) and Rappaport-Vassiliadis (RV) broths are employed after a 24-h non-selective enrichment to select for Salmonella and hamper the growth of competitive bacteria. In this study, we tested a new formulation of TT broth that lacks brilliant green dye and has lower levels of TT . We employed this TT broth formulation in conjunction with a 6-h non-selective pre-enrichment period and determined that Salmonella recovery was possible one day earlier than standard food culture methods. We tested the shortened culture method in different non-selective enrichment broths, enumerated Salmonella in the non-selective enrichments, and used 16S rRNA gene sequencing to determine the proportional abundances of Salmonella in the TT and RV selective enrichments. Together these data revealed that a 6-h non-selective pre-enrichment reduces the levels of competitive bacteria inoculated into the selective TT and RV broths, enabling the recovery of Salmonella 1 day earlier than standard culture enrichment methods.

Whole Genome DNA Sequence Analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS) to Salmonella subspecies enterica serotype Tennessee (S. Tennessee) to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana), which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP) analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs), suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks. Using WGS can delimit contamination sources for foodborne illnesses across multiple outbreaks and reveal otherwise undetected DNA sequence differences essential to the tracing of bacterial pathogens as they emerge.

Draft Genome Sequences of Nine Salmonella enterica Serovar Bovismorbificans Isolates from Various Sources.

The sequences of nine genomes of Salmonella enterica serovar Bovismorbificans were compared to study the diversity and distribution of this emerging virulent serovar. These whole-genome sequences fill some gaps in knowledge of the diversity of the isolates used in this investigation.

Whole-Genome Sequences of Six Salmonella enterica Serovar Bovismorbificans Isolates Associated with a 2011 Multistate Hummus-Borne Outbreak.

We present six draft genome sequences of Salmonella enterica serovar Bovismorbificans from isolates associated with the 2011 hummus-borne multistate outbreak. All six genome sequences indicate the presence of two plasmids, one of which demonstrates similarity to the 93-kb pSLT2 IncF-type plasmid of Salmonella enterica serovar Typhimurium.

Whole-Genome Sequencing of Salmonella enterica subsp. enterica Serovar Cubana Strains Isolated from Agricultural Sources.

We report the draft genomes of Salmonella enterica subsp. enterica serovar Cubana strain CVM42234, isolated from chick feed in 2012, and S. Cubana strain 76814, isolated from swine in 2004. The genome sizes are 4,975,046 and 4,936,251 bp, respectively.

Determination of the rat tissue partitioning of endotoxin in vitro for physiologically-based pharmacokinetic (PBPK) modeling.

The biosynthetically double-labeled lipopolysaccharide (LPS), containing (3)H-labeled on the fatty acyl-chains and (14)C-labeled on the glucosamine of Salmonella enterica serotype typhimurium, was isolated from bacteria grown in proteose peptone-beef extract (PPBE) medium in the presence of labeled precursors; 133 micro Ci/ml of [2-(3)H] acetate sodium salt and 0.167 micro Ci/ml of N-acetyl[D-1-(14)C]glucosamine. The LPS was extracted from the bacteria with 90% phenol/chloroform/petroleum ether, purified and stored in 0.1% (v/v) triethylamine/10 mM Tris HCl at -70 degrees C. Tissue slices and portions of the meninges were prepared and incubated in artificial cerebrospinal fluid (CSF) or Krebs phosphate buffer (Krebs) containing 150 ng/ml LPS with [(3)H] LPS (0.004 micro Ci/ml, sp. act. 28 micro Ci/mg LPS). The tissues were incubated under 95% oxygen/5% carbon dioxide at 37 degrees C with constant agitation until steady-state uptake was reached (60 min). At the end of the incubation period, tissues were processed for radioactivity measurement. The rat tissue partitioning of LPS in artificial CSF for brain and Krebs for other organs was measured by using the ratio of tissue to medium at the steady state in vitro. The following results were obtained from the study: Heart, 0.15; liver, 0.19; spleen, 0.12; kidney, 0.18; stomach, 0.17; small intestine, 0.18; brain stem, 0.10; cerebellum, 0.11; meninges, 0.77; hippocampus, 0.12; hypothalamus, 0.12; frontal cortex, 0.09 and caudate nucleus, 0.10. This information, along with plasma or blood/buffer partition coefficients, is a requisite for constructing a physiologically-based pharmacokinetic (PBPK) model of endotoxins for quantitative risk assessment.