Pathogens in 1 Gram of Ground Beef

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Open Access

Peer-reviewed

Research Article

Footing beef microbiome changes with antimicrobial decontamination interventions and product storage

• Margaret D. Weinroth,
• Brianna C. Britton,
• Kathryn R. McCullough,
• Jennifer North. Martin,
• Ifigenia Geornaras,
• Rob Knight,
• Keith E. Belk,
• Jessica L. Metcalf

10

• Published: June 5, 2019
• https://doi.org/10.1371/journal.pone.0217947

Figures

Abstract

Ground beef makes up more than than half of the beef consumed in the U.Southward. market. Although numerous studies take been conducted on microbial condom and shelf life of ground beefiness limited work has been done using a civilisation-independent approach. While past studies have allowed for the evaluation of a few organisms of involvement, in that location is limited piece of work on the microbial customs associated with fresh ground beefiness. In guild to accept a more complete picture of the microbial ecology of the product, a culture-independent approach utilizing 16S rRNA factor amplicon sequencing was used. The objectives of this report were to narrate the fresh footing beef microbiome and the effect that antimicrobial interventions and antioxidants, applied to beef trim before grinding, and production storage take on community limerick using 16S rRNA gene amplicon sequencing. Beef trimmings were treated with antimicrobials and an antioxidant. Samples were ground, loafed, and overwrapped before being packaged in modified-atmosphere packaging. Samples were in dark storage for 21 days followed by v days in retail display. Periodically during storage, samples were collected for microbiological analysis and DNA isolation. Due to low microbial biomass, but 52 of 210 samples were included in the final analysis. These samples represented 2 antimicrobial treatments (peroxyacetic acid, and a sulfuric acrid and sodium sulfate blend) and a control, from day-15 of nighttime storage and twenty-four hours-5 of retail display. As sample age increased, and so did the number of raw reads (P < 0.001) and aerobic plate counts (P < 0.001), which were correlated (r = 0.94, P = 0.017). Beyond all samples, lactic acrid bacteria were most abundant followed by Enterobacteriaceae; several rare taxa were also identified (namely Geobacillus, Thermus, and Sporosarcina). Antimicrobial treatment altered the bacterial alpha (P < 0.001) and beta (P = 0.001) diversity, while storage day altered alpha (P = 0.001) diversity. Enterobacteriaceae relative abundance differed (P < 0.05) among treatments and was highest in control samples. In addition to confirming previously described ascendant microbial differences in culture-dependent results, these data identified genera not typically associated with ground beef and allowed for study of shifts in the entire microbiome and non merely a subset of indicator organisms.

Citation: Weinroth MD, Britton BC, McCullough KR, Martin JN, Geornaras I, Knight R, et al. (2019) Ground beef microbiome changes with antimicrobial decontamination interventions and product storage. PLoS 1 14(6): e0217947. https://doi.org/ten.1371/journal.pone.0217947

Editor: Joy Scaria, Due south Dakota State University, Usa

Received: March two, 2019; Accepted: May 21, 2019; Published: June 5, 2019

Copyright: © 2019 Weinroth et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted utilise, distribution, and reproduction in any medium, provided the original writer and source are credited.

Information Availability: Samples described in this study have been deposited on QIITA, ID 10937 and EBI ERP113446.

Funding: The author(south) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Beef is a widely consumed protein in the U.S. marketplace, with an estimated per capita consumption of 54.iv lbs in 2017 [one]. Of this, an estimated 62% of all beef sold is basis [2]. The marketing of basis beef relies on a safe product that has an acceptable shelf life. In terms of prophylactic, Escherichia coli O157:H7, non-O157 Shiga toxin-producing Eastward. coli (STEC), and Salmonella spp. are pathogens that have been associated with footing beefiness [3–five]. In conjunction with safe concerns, the quality of ground beef over fourth dimension has also been extensively studied. While spoilage bacteria (such every bit Lactobacillus spp., Leuconostoc spp., and Pseudomonas spp. [6]) are not a threat to consumer wellness, their growth during product storage tin can decrease shelf life and increase off flavor and odors. Factors such as storage temperature, food safe interventions, packaging type, and length of distribution and retail brandish all have an result on the shelf life of the production.

E. coli O157:H7 and not-O157 STECs are adulterants in basis beef according to the U.S. Department of Agriculture Nutrient Prophylactic and Inspection Service (USDA-FSIS), significant in that location is zero-tolerance for their presence in the product [7]. On the other hand, Salmonella spp. is governed past a performance standard which sets a maximum threshold allowed in ground beef [eight]. The prevalence of these pathogen groups can be reduced through the utilize of chemic and/or concrete decontamination interventions during the slaughter process [9]. Chemic treatments of the meat, such equally lactic acid, peroxyacetic acrid, and a commercial alloy of sulfuric acrid and sodium sulfate, have been shown to reduce pathogen contamination on beef products through diverse methods of application at different stages of processing [10–13].

The shelf life of footing beef tin too be extended with the use of chemical interventions, because these interventions do not simply target pathogenic leaner but those involved in spoilage as well. However, although chemic interventions may reduce spoilage leaner numbers, these interventions may also impact quality attributes that greatly affect consumer preference, such as color [14]. As a result, in that location must exist a balance to the number and type of interventions used that maximizes both safety and quality. In addition to chemic interventions, antioxidants, sometimes used in tandem with chemical interventions, reduce oxidation of lipids and proteins, increasing shelf life [15]. The effect of these interventions on the fresh meat microbiome is unknown.

Another component of the safety and quality of basis beef is packaging. Modified atmosphere packaging (MAP) is a system of packaging that alters the gaseous components of a package assuasive for the extended shelf life of a production [16]. Components of MAP packaging oft include carbon monoxide (CO), carbon dioxide (CO_ii), and oxygen at different levels. These are combined to maintain color throughout storage, while minimizing microbial growth and lipid oxidation [14]. In add-on to increasing shelf life, MAP packaging inhibits pathogen growth, including E. coli O157:H7 [17] and spoilage organisms [xviii]. In depression oxygen mixtures, these changes in temper probable affect the microbial community associated with fresh meat past selecting for anaerobes or facultative anaerobes.

The efficacy of interventions and packaging technologies in reducing microbial numbers and inhibiting growth during product storage, respectively, has been primarily studied using traditional culture-based methods. While this approach is useful in understanding the changes to a targeted organism of interest, culturing approaches do not capture changes in the unculturable component of the microbiome. Characterizing the full diversity of the bacterial community can provide insights into ecologies that may support or non back up the organism of involvement, and how the microbial community changes with the application of antimicrobial interventions and subsequent product storage. In order to evaluate these bacterial community changes, sequencing of the 16S rRNA factor tin be used to quantify a relative abundance of bacterial taxa present. In terms of the fresh beef microbiome, limited work has been done using culture-independent methods [19,xx]. For example, piece of work has been done on footing beef specific to 16S rRNA gene sequencing of cultured lactic acrid bacteria isolates [20], while another 16S rRNA gene sequencing written report was conducted on whole muscle beef cuts rather than ground product [19]. With this in mind, the objectives of this written report were to narrate the fresh basis beef microbiome and the effect of antimicrobial interventions, antioxidants, and storage on this community using 16S rRNA gene amplicon sequencing.

Materials and methods

Sample drove and antimicrobial treatment

Basis beef trimmings (80% lean) were collected from a commercial harvest facility, transported to Colorado Land University (Fort Collins, CO) within one h of collection, and stored overnight at iv°C. The side by side day, the trim was divided into v different ninety kg batches for subsequent antimicrobial handling using a custom-built spray cabinet (Birko/Chad Equipment, Olathe, KS) designed for trim and sub-central cuts. The chiffonier was fitted with 18 floodjet spray nozzles (0.38 liter/min; Grainger Industrial Supplies, Lake Forest, IL), with ten nozzles affixed above the product chugalug and eight nozzles below. Antimicrobial treatments selected for inclusion in the study are used by the beef manufacture and their antimicrobial effects against pathogen contamination on beefiness products has previously been reported [10, 12]. The antimicrobial treatments evaluated were: (1) untreated control, (2) lactic acid (iv%, LA; Corbion, Lenexa, KS), (three) a commercially bachelor blend of sulfuric acrid and sodium sulfate (pH 1.2, SASS; Zoetis, Parsippany, NJ), (iv) peroxyacetic acid (350 ppm, PAA; Kroff Food Service, Inc., Pittsburgh, PA), and (v) PAA (350 ppm) acidified with SASS (pH one.ii, PAA/SASS). Spray treatments were applied at a pressure level of 0.15 MPa with a product contact time of 10 to 11 s. Untreated (command) and treated batches of trim were then stored at iv°C for 24 h prior to further processing.

Grinding, antioxidant application, storage, and display

Trim treatments were separately course footing to 12.7 mm, and afterward the class grind, each treatment was divided in one-half. Forty-five kilograms of each treatment was retained for antioxidant application while the other one-half was fine ground to three.2 mm. For antioxidant application, 224.2 one thousand (0.494% of the total weight) of a commercially prepared antioxidant (dried vinegar and natural flavors, Wenda Ingredients, Prosur, Murica, Spain) at a 0.iii% concentration was added following the initial grade grind and mixed for 30 s using a countertop mixer (KitchenAid, Benton Harbor, MI), followed by a fine grind to three.two mm. Immediately after grinding, a sample from each antimicrobial/antioxidant combination was retained for microbiological analysis and sequencing. All production was loafed by hand into 450 g loaves and placed onto 2P trays (Team Packaging Inc., Denver, CO) with an absorbent pad and individually overwrapped with a gas permeable plastic film.

Batches of five trays of ground beef of the same treatment were placed into private 50 × 50 cm modified atmosphere packaging (MAP) bags (SealedAir, Denver, CO), with an oxygen scavenger. The bags were sealed using a MAP packager (Corr-Vac; G-Tek, Elgin, IL) to remove the oxygen and fill up the bags with a Tri-Gas mixture (19.vi% CO₂, 0.four% CO, and balancing N; Airgas, Salt Lake City, UT) prior to sealing. Numberless were so placed in opaque cardboard boxes, sealed, and stacked in 4°C for upwardly to 21 days in night storage to emulate common industry practices for storage times. Following the 21-twenty-four hour period storage flow, the trays of basis beef were removed from the MAP arrangement and were placed in a simulated retail display case (four°C) for upwards to five days (once more to emulate common industry practices). Samples were nerveless for microbiological analysis and sequencing on days 0, half dozen, and fifteen of dark storage, and on day 5 of retail display after 21 days of night storage. It should be noted that additional samples were placed into dark storage and retail display for another study not described in the present work, thus the surplus of ground beef loaves.

Microbiological assay

At each sampling time, 10 samples from each of the ten basis beef treatments (i.e., five antimicrobial treatments, with and without the add-on of the antioxidant) were analyzed for aerobic plate counts (APC). For the analysis, a l-one thousand portion of each sample was aseptically transferred to a filter Whirl-Pak purse, to which 100 mL of Dey-Engley neutralizing broth (Difco, Becton Dickinson [BD], Sparks, Medico) was added. Samples were mechanically pummeled for ii min and and so serially-diluted in 0.1% buffered peptone water (Difco, BD). Appropriate dilutions were plated, in indistinguishable, onto Petrifilm Aerobic Plate Count plates (3M, St. Paul, MN) and colonies were counted afterward incubation of plates at 35°C for 48 h.

16S rRNA cistron amplicon sequencing

Dry sterile swabs (Becton Dickinson and Co., Sparks, MD) were used to collect Dna from the same ground beef samples that were analyzed for APC (in addition to a sample immediately afterward fine grinding). Sampling was performed by inserting the swab into the geometric eye of the loaf and rotating the swab several times. After sampling, swabs were returned to their original holder and immediately placed at -xx°C before transportation to the Academy of Colorado-Boulder for Dna extraction. DNA was extracted and processed 1 to ii months mail service collection. DNA extraction and sequencing library preparation followed the Earth Microbiome Project standard protocols [21]. Briefly, DNA was extracted from swabs using the MoBio Powersoil 96-well kit (Mo Bio Laboratories, Inc., Carlsbad, California) with the 515F-Y with barcode/806R primer ready for amplification of the V4 region [21–23]. 16S rRNA amplicons were sequenced on an Illumina MiSeq at the University of California-San Diego, Found for Genomic Medicine (150x1).

Bioinformatics and statistics

Amplicon sequences were candy through the QIIME2 (2018.half dozen) pipeline [24]. Samples were demultiplexed and assigned exact sequence variance (ESV) using the DADA2 plugin [25] with no truncation of the reads and the chimera method set up to pooled. Multiple sequence alignment of the sequences was completed with MAFFT [26] and filtered to remove highly variable positions. FastTree two [27] was used to construct and root a phylogenetic tree. Taxonomic classification was conducted using a pretrained Naive Bayes classifier trained on the Greengenes database for the 16S rRNA region spanning 515/806 region [28]. Reads assigned to mitochondria and chloroplast were removed from downstream analysis. Samples were rarified at a depth of 8021, allowing retention of 52 samples. This rarefication depth was chosen to maintain every bit many samples every bit possible while retaining as much sequence depth as possible; there was a decrease in sequencing depth to 3506 afterward the sample containing 8021.

Alpha diversity was assessed via Faith'southward Phylogenetic Variety and beta diverseness was measured using weighted UniFrac distances (unweighted results found in S1 Fig). Alpha diversity, after verification of a normal distribution using the 'shapiro.test' function, was compared using the lm and anova functions in R version 3.5.ane and compared using the CLD function from the emmeans package. Beta diversity interactions and main effects were evaluated using the adonis function from Vegan (v. ii.five–two). Differential affluence was conducted at the aggregated family unit level via ANCOM [29]. In all comparisons, α = 0.05 and an FDR adjustment was used when appropriate.

Colony counts recovered from the Petrifilm Aerobic Count plates were converted to log CFU/k. While there was an interaction present between sampling twenty-four hour period and treatment, equally determined past the 'glm' and 'emmeans' functions in base of operations R (v. 3.5.one) and emmeans package (v. 1.2.iii), the treatments were pooled across day for the comparison of sequenced reads to APC. The APCs were averaged to obtain an APC per 24-hour interval of the written report, and the same was done for raw sequences. The 'cor.examination' role was used with default settings to determine if there was a correlation between number of reads sequenced and APC by sampling day.

Accession number

Samples described in this written report take been deposited on QIITA, ID 10937 and EBI ERP113446.

Results and discussion

Full general sequencing results

Sequencing of the 16S rRNA V4 partial cistron region of 210 DNA samples isolated from swabs of basis beef (and 31 bare swabs) generated 1.6M reads with an average of 7391 reads per sample (range 0 to 53708). Quality filtering resulted in removal of one.5% of reads across all samples. Of remaining filtered reads, 0.96% were classified as chimeric and removed. Sequencing depth was considered advisable via the construction of a rarefaction bend (S2 Fig). Due to the low biomass of bacteria and subsequent low number of sequenced reads establish within specific storage days and antimicrobial treatments, only 52 samples were included for downstream analysis (Table 1). Of the blank sample swabs, 11 sequenced with an average read count of just 12 reads.

Culture results agree with low biomass sequencing results

Ground beef sampled at later times during the study generated a greater (P > 0.001) number of reads per sample (Fig 1), regardless of antimicrobial handling. Aerobic plate counts (APC) likewise increased (P > 0.001) by day of sampling (Fig 1). In fact, the number of read sequences and APC counts were highly correlated (r = 0.94, P = 0.017). The increase of culturable bacteria during retail display has been previously documented. Brooks et al. [30] observed an increase in psychrophilic APC in low-oxygen MAP packaging over a 21 solar day retail display period while Lavieri and Williams [31] reported a similar trend in regards to ground beefiness during storage. The correlation between the civilisation-independent and culture arroyo demonstrates that early on in basis beef storage, the microbial customs biomass is very depression.

Fig 1. Raw reads sequenced versus aerobic plate counts past day.

Number of raw reads sequenced per storage day of the study compared to aerobic plate counts (APC) recovered from the aforementioned samples on the same twenty-four hours. In that location was an increase (P < 0.05) in both raw reads and APC populations over the duration of the study. These ii measurements were institute to be correlated to each other (r = 0.94, P = 0.017).

https://doi.org/10.1371/journal.pone.0217947.g001

Antioxidants had no result on the basis beefiness microbiome

In this study, some of the antimicrobial interventions had antioxidants added (S1 Table). Even so, it was plant in that location was no difference in alpha (P = 0.79) or beta diversity (P = 0.38) between footing beefiness microbiomes treated and not treated with the antioxidants. As a upshot, this variable was not considered in downstream assay.

Firmicutes dominated the microbiome, regardless of storage day or antimicrobial handling

Firmicutes was the boss phylum of bacteria across all storage days and antimicrobial interventions, comprising 70 to 99.ix% (with a hateful of 97.iii%) of bacterial relative abundance. Bacterial taxa in the order Lactobacillales (LAB) were dominant inside the Firmicutes phylum and included families such every bit Leuconostocaceae, and Lactobacillaceae (Fig 2). The high level of LAB was not unexpected due to the use of packaging with low oxygen, and in conjunction with CO₂, which promotes LAB growth [32]. Anaerobically packaged ground beef has been previously reported to be dominated by LAB in culture studies [6] and LAB isolation followed past 16S rRNA characterization [20].

Proteobacteria was the 2nd most common phylum represented in the beefiness microbiome (Fig 2). Within Proteobacteria, the family unit Enterobacteriaceae accounted for the greatest portion of the phylum followed by Pseudomonadaceae. The Enterobacteriaceae family unit (which made up one.75% of the total microbiome) encompasses a number of pathogens of interest in meat production, such as Salmonella spp., E. coli O157:H7, and non-O157 Shiga toxin producing E. coli (STEC); thus, Enterobacteriaceae is commonly used as an indicator organism for potential presence of pathogens. The rationale behind an indicator organism is that if there is a reduction of the family of bacteria, the pathogenic sub-population also will reject in magnitude appropriately [33]; though some studies have shown just limited direct relationships between indicators and pathogens [34]. The depression levels of Pseudomonadaceae and Streptococcaceae found in the samples were not surprising as these bacterial families have previously been associated with spoilage of meat stored nether refrigeration atmospheric condition [6, 35] (though in the case of Pseudomonadaceae it has been prominently associated with aerobic packaging conditions [36]).

In addition to dominant leaner, rare taxa were identified

While LAB and Enterobacteriaceae were found to contain the bulk of the bacterial community, three genera were as well institute in more half of the samples: Geobacillus (mean = 0.07%; range 0.00 to 0.70%), Thermus (hateful = 0.04%; range 0.00 to 0.32%), and Sporosarcina (hateful = 0.06%; range 0.00 to 0.19%). Within extraction blanks, Sporosarcina had one striking beyond all blanks while the other two genera did not take any reads assigned to them; indicating these genera are likely the event of a biological presence, not laboratory or reagent contamination. While Geobacillus is an aerobic thermophile, it has been isolated from many libation environments, such as absurd soils and ocean water; which has been attributed to the longevity and mobility of this genus in the atmosphere [37]. Recently, Geobacillus stearothermophilus was isolated from imported frozen beefiness meat collected in Cairo, Arab republic of egypt [38]. The genus Thermus is anaerobic and heat resistant, and has been found in many environments including canned meat, soil, animal feces and dust [39]. Finally, Sporosarcina, are gram positive strict aerobes [40] that are considered important spore-formers in meat systems [41] and have been constitute on meat contact surfaces in Islamic republic of pakistan [42]. An interesting characteristic these genera share is their characterization every bit extremophiles or spore formers.

Antimicrobial intervention and sampling solar day independently acted on the microbiome

Before being evaluated individually, the interaction betwixt the antimicrobial treatment practical to the beef trim and ground beefiness storage twenty-four hour period was considered. The antimicrobial treatment by sampling mean solar day interaction did not affect (P = 0.336) overall customs composition. Likewise, alpha diversity was not impacted (P = 0.703) by an interaction between antimicrobial treatment and sampling day.

Antimicrobial treatment alters the microbiome and lowered Enterobacteriaceae

Handling of beef trimmings with peroxyacetic acid (PAA) or the sulfuric acid and sodium sulfate blend (SASS) prior to grinding, contradistinct many aspects of the beef microbiome. When beta multifariousness was compared, antimicrobial treatments differed (P = 0.001, Fig 3a) from each other. This was besides true in the case of blastoff diversity (P < 0.001, Fig 3b) which was found to exist lower in the SASS samples when compared to the control and PAA samples. Both chemical interventions appeared to cluster together and change beta multifariousness in the same style, though SASS had an affect (P < 0.001) on alpha diversity while PAA did not. Three families of bacteria were found to differ (P < 0.05) in relative abundance across treatments: Enterobacteriaceae, Lactobacillaceae, and Leuconostocaceae (Fig 4). The average occurrence of Enterobacteriaceae was 4.vii% (range 0.1 to 25.ane%) in untreated control samples as compared to the SSAS (average = 0.i%, range 0.0 to 0.4%) and PAA (average = 0.2%, range 0.0 to 0.7%). The higher percentage associated with Enterobacteriaceae in the ground beefiness non treated with a chemic intervention suggests the chemical treatments are appropriate for decreasing Enterobacteriaceae on ground beef product. Enterobacteriaceae reduction as a result of interventions has previously been demonstrated throughout the beef slaughter process [43]. In previous culture-independent footing beef work [20], Leuconostoc spp., from the family unit Leuconostocaceae was found to be the dominant LAB organism. Interestingly, in the current study, Leuconostocaceae was found in a lower abundance in samples without chemical intervention (average = 22.7%, range iv.3 to 41.0%) when compared to the SASS (average = 48.8%, range 34.six to 65.two%) and PAA (average = 46.ix%, range vii.7 to 88.two%) treatments. By antimicrobial intervention piece of work has demonstrated that gram negative bacteria are typically more sensitive than gram positive bacteria when treated with an antimicrobial, such as a weak organic acid [44], due to differences in prison cell wall composition. So, while it was not a surprise to see such a loftier relative abundance of LAB (because they are gram positive), different LAB composition found within different chemical intervention samples may indicate that some families of LAB are more sensitive to specific chemical intervention than others. Another cistron to consider, specific to the relative abundance of Lactobacillaceae between SASS and PAA treated ground beef, is that these differences could stalk from differing pH acidities of SASS and PAA (SASS had a lower pH) that would in turn have contradistinct the footing beef pH and LAB populations.

Fig iii. Ground beefiness treated with dissimilar antimicrobial treatments had different blastoff and beta variety.

Effect of antimicrobial treatment of beef trimmings on ground beef microbiomes beyond nighttime storage and retail display days. (a) Full bacterial community differences between different treatments as measured by weighted Unifrac Distances were different (P = 0.001) and (b) alpha diversity (P < 0.001), as measured by Faith's phylogenic diversity.

https://doi.org/10.1371/periodical.pone.0217947.g003

Fig 4. Iii bacterial families differed between antimicrobial treatments: Enterobacteriaceae, Lactobacillaceae, and Leuconostocaceae.

When analysis of composition of microbiomes (ANCOM) was conducted at the family level, eleven families were compared. 3 of these families were considered significantly unlike: (a) Enterobacteriaceae, (b) Lactobacillaceae, and (c) Leuconostocaceae. The Due west statistic represents the number of pairwise comparisons that were considered significantly different. In this written report, eleven families were identified across all samples.

https://doi.org/10.1371/journal.pone.0217947.g004

While a direct comparison between conventional civilisation methods and amplicon methods was non the intention of this study, these data provide some insight into future applications of the amplicon methodology in answering meat science questions. Identification across the family unit or genus level of bacteria was not the intention or possible with the molecular methods employed hither. While other molecular methods (such as shotgun metagenomics or quantitative PCR) could provide a higher level of phylogenetic clarity, these data were intended to provide an overview of the ecological shift due to treatments—which has been demonstrated by the observed shifts in Enterobacteriaceae, Lactobacillaceae, and Leuconostocaceae populations. If the goal was to measure the reduction of specific pathogens without the wider context of other shifts in the microbiome, culturing on selective media or other molecular methods would have been more advisable.

Alpha diversity decreased over time

While beta diversity did not differ (P = 0.395) between day-xv of night storage and twenty-four hour period-v of retail display, alpha diversity did differ (P = 0.001; Fig 5). Day-15 of dark storage had an boilerplate Religion's phylogenetic diverseness (FPD) of 7.57 (95% C.I. 6.93 to 8.xx) while day-5 of retail brandish had an average FPD of 5.80 (95% C.I. 5.ten to six.51). This finding shows a decrease in diversity paired with an increment in bacterial growth over the ii fourth dimension points. In other words, although biomass of bacteria increased over fourth dimension, the community included fewer types. The two storage days had unlike (P = 0.04) boilerplate numbers of verbal sequence variants (ESV) present; day-xv of dark storage had, on average, 21 ESVs nowadays (95% C. I. 18 to 24), while mean solar day-5 of retail display had xvi ESVs present (95% C. I. 12 to 19). This could exist due to LAB out competing other bacterial taxa over storage fourth dimension. An increase in the blastoff diversity of LAB has been associated with MAP packaging when compared to non-MAP, though non over duration of storage [twenty]. At the same time, Brooks et al. [30] observed an increase in LAB over retail display while Lavieri and Williams [31] observed the same increase in ground beef in storage. Though there was a numeric increase in relative abundance of LAB over fourth dimension (1.3% on the dark storage day versus 2.3% on the retail brandish day), this difference was non pregnant (P > 0.05), demonstrating 1 of the challenges associated with amplicon sequencing data. Specifically, relative abundance is not absolute abundance inside a community; if the LAB were present in 90% of a sample but the unabridged customs was 2 logs lower than a comparison, we may non run across a log fold deviation. Still, 16S rRNA cistron sequencing does allow a level of description of the community non possible with traditional culture techniques, making the two approaches highly complementary.

Fig 5. Between days of experiment, beta diversity was not different just alpha multifariousness did differ.

Differences between storage days on ground beef microbiomes across beefiness trim chemical interventions. (a) Total bacterial customs differences betwixt days, as measured by weighted Unifrac Distances, were non different (P = 0.395) but (b) alpha diverseness, as measured by Faith's phylogenic diversity, was unlike (P = 0.001).

https://doi.org/10.1371/periodical.pone.0217947.g005

Conclusions

These data allowed for the characterization of the fresh ground beef microbiome and effects of common practices of chemical interventions and packaging on the product. Results showed that Firmicutes, mainly Lactobacillales (a known spoilage organism in ground beef), dominated the microbiome across all treatments while several rare genera of extremophiles or spore formers were characterized that are non typically associated with ground beef spoilage. Furthermore, Enterobacteriaceae was reduced equally a result of antimicrobial treatment, which is consistent with civilization data. I claiming of this study was the low biomass during the early on days of nighttime storage. As a result, many of the samples on the day of grinding and in the early on days of dark storage did not sequence at a sufficient depth to be included in the analysis. This event was supported by APC, which were besides lower in early days of dark storage when compared to sample days later in the report. Overall, these data provide a different look at common shelf life and nutrient prophylactic practices in ground beef, allowing for both confirmation of ascendant phyla and descriptions of lesser reported bacteria for a higher level of clarity into the changes in the microbiome, non afforded to u.s. by culture-based methods solitary.

Supporting information

S1 Fig. Unweighted UniFrac showed treatment but non day differences.

Total bacterial community differences between dissimilar days (A) and treatments (B) as measured by unweighted Unifrac Distances. Day was non considered significant (P = 0.071) while treatment was (P = 0.009).

https://doi.org/10.1371/periodical.pone.0217947.s001

(TIF)

Acknowledgments

The authors would like to give thanks Zoetis for allowing us to obtain our sample from an ongoing shelf-life project and Colorado State University for support related to Dna sequencing. Additionally, thank you to all of the CSU Meat Science graduate students that assisted with sampling.

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Source: https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0217947