i need help finding the the type of metagenomic data that the study gathered in the article and explain what they did and the data

https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-018-1323-4
Transcribed Image Text:Metagenomics of pasteurized and unpasteurized gouda cheese using targeted 16S rDNA sequencing Joelle K. Salazar¹, Christina K. Carstens¹, Padmini Ramachandran², Arlette G. Shazer¹, Sartaj S. Narula³, Elizabeth Reed², Andrea Ottesen² and Kristin M. Schill¹* Abstract Background: The microbiome of cheese is diverse, even within a variety. The metagenomics of cheese is dependent on a vast array of biotic and abiotic factors. Biotic factors include the population of microbiota and their resulting cellular metabolism. Abiotic factors, including the pH, water activity, fat, salt, and moisture content of the cheese matrix, as well as environmental conditions (temperature, humidity, and location of aging), influence the biotic factors. This study assessed the metagenomics of commercial Gouda cheese prepared using pasteurized or unpasteurized cow milk or pasteurized goat milk via 16S rDNA sequencing. Results: Results were analyzed and compared based on milk pasteurization and source, spatial variability (core, outer, and under the rind), and length of aging (2-4 up to 12-18 months). The dominant organisms in the Gouda cheeses, based on percentage of sequence reads identified at the family or genus levels, were Bacillaceae, Lactococcus, Lactobacillus, Streptococcus, and Staphylococcus. More genus- or family-level (e.g. Bacillaceae) identifications were observed in the Gouda cheeses prepared with unpasteurized cow milk (120) compared with those prepared with pasteurized cow milk (92). When assessing influence of spatial variability on the metagenomics of the cheese, more pronounced differences in bacterial genera were observed in the samples taken under the rind; Brachybacterium, Pseudoalteromonas, Yersinia, Klebsiella, and Weissella were only detected in these samples. Lastly, the aging length of the cheese greatly influenced the number of organisms observed. Twenty-seven additional genus-level identifications were observed in Gouda cheese aged for 12-18 months compared with cheese only aged 2-4 months. Conclusions: Collectively, the results of this study are important in determining the typical microbiota associated with Gouda cheese and how the microbiome plays a role in safety and quality. Keywords: Metagenome, 16 s rDNA, Cheese, Dairy, Gouda, Unpasteurized milk Background High-throughput metagenomic sequencing technology has transformed the ecological study of food products. Targeted metagenomics utilizes gene fragment DNA se- quencing to determine identities of microbiota such as bacteria, yeast and mold. In most cases, a conserved seg- ment of a hypervariable region of the 16S rDNA gene is used. In recent years, the field of metagenomics has * Correspondence: kristin.schill@fda.hhs.gov Division of Processing ence and Technology, U.S. Food and Drug Administration, Bedford Park, IL, USA Full list of author information is available at the end of the article BMC CrossMark of Safety, flourished and published studies now include insights into the microbiomes of cilantro [1], spinach [2], bean sprouts [3], kimchi [4], kefir [5], meat [6], wine [7], and cheese [8-13]. The composition of the native microbiota in these food products may help determine property characteristics contributed to by microorganisms such as flavor, texture, color, aroma, shelf-life, and spoilage. Cheese is composed of microorganisms, originating from the raw ingredients used, the environment, and added starter cultures as well as adjunct cultures. These many sources of microbes cause considerable variability in the microbiome across cheese varieties. Ⓒ The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Transcribed Image Text:in this family are often harder to eliminate in the process- ing environment due to their ability to form biofilms and heat-resistant endospores permitting their resilience to sanitization processes. Staphylococcus reads were found in relatively low numbers in the Gouda cheeses (2.0-13.4%). Staphylococ- cus species, such as Staphylococcus equorum, can be used as an additive to the starter culture for certain semi-hard cheeses such as Swiss cheese [37] and are also naturally occurring microorganisms in cheese brines. Interestingly, it has been determined that S. equorum possesses anti-listeria properties and some studies have suggested the use of this species as a protective starter culture [38]. Staphylococcus has been detected at 0.17% in semi-hard cheeses [8], and at less than 3% in Latin-style cheeses [9]. Staphylococcus has been found in high numbers (5-25%) on the surface of certain cheeses, especially early in the aging process and in cheeses made using goat milk [20, 39]. Staphylococcus species epidermidis and caprae, have also been isolated from goat milk [40]. There were a greater number of genus- or family-level identifications observed for the pasteurized goat (n = 138) and unpasteurized cow Gouda cheeses (n=120) com- pared with the pasteurized cow Gouda cheeses (n = 92). This is not surprising, as unpasteurized milk has not undergone treatment to eliminate pathogens and re- duce the bacterial burden. This is consistent with other studies that have shown unpasteurized cheeses contained a more diverse microbiome than pasteur- ized cheeses [41]. In this study, 18 genera were iden- tified only in unpasteurized cow Gouda cheese and not in the pasteurized cow or goat Gouda cheeses. Some of the genera identified in the unpasteurized Gouda included Mycoplasma, Ochrobactrum, Nocar- dioides, Yaniella, and Adhaeribacter. Mycoplasma is a bacterium that can cause mastitis in dairy cattle, and Ochrobactrum has been isolated from cow teat skin [42]. Nocardioides, Yaniella, and Adhaeribacter have all previously been identified in unpasteurized milk and cheese [23, 43, 44]; Yaniella, a Gram positive coccus in the family Micrococcaceae, has been typic- ally found in saline soils and has also been found in cheese rinds [23]. Eight bacterial genera were identified only in pasteur- ized cow Gouda cheese and included Anoxybacillus, Curtobacterium, and Yersinia. All three genera have pre- viously been isolated from dairy products [45-48]. Anoxy- bacillus is a thermophilic spore-former frequently isolated from whole milk powder and nonfat dry milk and is some- times used as a hygiene indicator in pasteurized diary manufacture due to its high optimum growth temperature [48]. Unpasteurized milk often contains the potential pathogen Yersinia enterocolitica and the organism can be found in curd samples when the milk is used to make cheese. However, in one study, Y. enterocolitica has been identified in one out of 265 pasteurized milk samples [47]. A total of 28 genera were identified only in the pasteur- ized goat Gouda cheese in this study and included Mannheimia, Leptotrichia, Balneimonas, Klebsiella, and Pseudoalteromonas. Mannheimia and Leptotrichia may have been part of the goat ecosystem which was transmit- ted to the milk used in the manufacture of the cheeses. The genus Mannheimia is comprised of bacteria respon- sible for epizootic pneumonia and mastitis in goats, sheep, and cattle [49], while Leptotrichia has been isolated from goat foot lesions [50] and are normally found in the hu- man oral cavity [51]. Balneimonas has not previously been isolated from cheese, but has been isolated from Suanzhou (Chinese fermented cereal gruel) samples [52]. Bacteria in the genus Klebsiella, such as Klebsiella pneumoniae, are human pathogens and have also been found to cause spoilage in cheese via gas production leading to early blowing of semi-hard and hard cheeses [53]. Like many other organisms, Klebsiella lack thermoresistance, which indicates con- tamination of the cheeses most likely occurred during or post-manufacture. Pseudoalteromonas are mesophi- lic or psychrophilic marine bacteria that can survive in environments with high salinity. The genera has been identified in soft and semi-hard cow pasteurized and unpasteurized cheeses as well as in cheese rinds [8] and on the surfaces of smear-ripened cheeses [54, 55]. In one study, Pseudoalteromonas haloplanktis comprised 17% of the total mapped reads of a smear-ripened cheese as determined using 16S rDNA metagenomics sequencing [54]. Overall, the majority of all the bacterial genera identi- fied in this study were present in all three cheese regions (under the rind, core, inside), however differences were observed in population proportions. In the samples taken under the rind, Staphylococcus and Tetragenococ- cus were prevalent (9.6 and 4.8% of the total sequencing reads for all cheeses, respectively). Large populations of Staphylococcus on the surfaces of cheeses have been de- tected previously [23, 56]. Tetragenococcus, a moderately halophilic bacterial genus, has previously been detected in unpasteurized hard cheeses and cheese rinds at 0.05 and 0.18%, respectively, but was not detected in soft or semi-hard cheeses [8]. In addition, Brachybacterium, Pseudoalteromonas, Yersinia, Klebsiella, and Weissella were only detected in the under the rind samples. Brachybacterium and Pseudoalteromonas are both halo- philes, capable of growing in concentrations of salt as high as 15-18% [57, 58]. Therefore, these organisms may have contaminated the cheese during brining. Yersinia and Klebsiella contain species which are poten- tial human pathogens and are ubiquitous in the

Question

i need help finding the the type of metagenomic data that the study gathered in the article and explain what they did and the data

https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-018-1323-4

Metagenomics of pasteurized and
unpasteurized
gouda cheese using
targeted 16S rDNA sequencing
Joelle K. Salazar¹, Christina K. Carstens¹, Padmini Ramachandran², Arlette G. Shazer¹, Sartaj S. Narula³,
Elizabeth Reed², Andrea Ottesen² and Kristin M. Schill¹*
Abstract
Background: The microbiome of cheese is diverse, even within a variety. The metagenomics of cheese is
dependent on a vast array of biotic and abiotic factors. Biotic factors include the population of microbiota and their
resulting cellular metabolism. Abiotic factors, including the pH, water activity, fat, salt, and moisture content of the
cheese matrix, as well as environmental conditions (temperature, humidity, and location of aging), influence the
biotic factors. This study assessed the metagenomics of commercial Gouda cheese prepared using pasteurized or
unpasteurized cow milk or pasteurized goat milk via 16S rDNA sequencing.
Results: Results were analyzed and compared based on milk pasteurization and source, spatial variability (core,
outer, and under the rind), and length of aging (2-4 up to 12-18 months). The dominant organisms in the Gouda
cheeses, based on percentage of sequence reads identified at the family or genus levels, were Bacillaceae,
Lactococcus, Lactobacillus, Streptococcus, and Staphylococcus. More genus- or family-level (e.g. Bacillaceae)
identifications were observed in the Gouda cheeses prepared with unpasteurized cow milk (120) compared with
those prepared with pasteurized cow milk (92). When assessing influence of spatial variability on the metagenomics
of the cheese, more pronounced differences in bacterial genera were observed in the samples taken under the
rind; Brachybacterium, Pseudoalteromonas, Yersinia, Klebsiella, and Weissella were only detected in these samples.
Lastly, the aging length of the cheese greatly influenced the number of organisms observed. Twenty-seven
additional genus-level identifications were observed in Gouda cheese aged for 12-18 months compared with
cheese only aged 2-4 months.
Conclusions: Collectively, the results of this study are important in determining the typical microbiota associated
with Gouda cheese and how the microbiome plays a role in safety and quality.
Keywords: Metagenome, 16 s rDNA, Cheese, Dairy, Gouda, Unpasteurized milk
Background
High-throughput metagenomic sequencing technology
has transformed the ecological study of food products.
Targeted metagenomics utilizes gene fragment DNA se-
quencing to determine identities of microbiota such as
bacteria, yeast and mold. In most cases, a conserved seg-
ment of a hypervariable region of the 16S rDNA gene is
used. In recent years, the field of metagenomics has
* Correspondence: kristin.schill@fda.hhs.gov
Division of Processing ence and Technology,
U.S. Food and Drug Administration, Bedford Park, IL, USA
Full list of author information is available at the end of the article
BMC
CrossMark
of
Safety,
flourished and published studies now include insights
into the microbiomes of cilantro [1], spinach [2], bean
sprouts [3], kimchi [4], kefir [5], meat [6], wine [7], and
cheese [8-13]. The composition of the native microbiota
in these food products may help determine property
characteristics contributed to by microorganisms such as
flavor, texture, color, aroma, shelf-life, and spoilage.
Cheese is composed of microorganisms, originating
from the raw ingredients used, the environment, and
added starter cultures as well as adjunct cultures.
These many sources of microbes cause considerable
variability in the microbiome across cheese varieties.
Ⓒ The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

in this family are often harder to eliminate in the process-
ing environment due to their ability to form biofilms and
heat-resistant endospores permitting their resilience to
sanitization processes.
Staphylococcus reads were found in relatively low
numbers in the Gouda cheeses (2.0-13.4%). Staphylococ-
cus species, such as Staphylococcus equorum, can be
used as an additive to the starter culture for certain
semi-hard cheeses such as Swiss cheese [37] and are also
naturally occurring microorganisms in cheese brines.
Interestingly, it has been determined that S. equorum
possesses anti-listeria properties and some studies have
suggested the use of this species as a protective starter
culture [38]. Staphylococcus has been detected at
0.17% in semi-hard cheeses [8], and at less than 3%
in Latin-style cheeses [9]. Staphylococcus has been
found in high numbers (5-25%) on the surface of
certain cheeses, especially early in the aging process
and in cheeses made using goat milk [20, 39].
Staphylococcus species epidermidis and caprae, have
also been isolated from goat milk [40].
There were a greater number of genus- or family-level
identifications observed for the pasteurized goat (n = 138)
and unpasteurized cow Gouda cheeses (n=120) com-
pared with the pasteurized cow Gouda cheeses (n = 92).
This is not surprising, as unpasteurized milk has not
undergone treatment to eliminate pathogens and re-
duce the bacterial burden. This is consistent with
other studies that have shown unpasteurized cheeses
contained a more diverse microbiome than pasteur-
ized cheeses [41]. In this study, 18 genera were iden-
tified only in unpasteurized cow Gouda cheese and
not in the pasteurized cow or goat Gouda cheeses.
Some of the genera identified in the unpasteurized
Gouda included Mycoplasma, Ochrobactrum, Nocar-
dioides, Yaniella, and Adhaeribacter. Mycoplasma is a
bacterium that can cause mastitis in dairy cattle, and
Ochrobactrum has been isolated from cow teat skin
[42]. Nocardioides, Yaniella, and Adhaeribacter have
all previously been identified in unpasteurized milk
and cheese [23, 43, 44]; Yaniella, a Gram positive
coccus in the family Micrococcaceae, has been typic-
ally found in saline soils and has also been found in
cheese rinds [23].
Eight bacterial genera were identified only in pasteur-
ized cow Gouda cheese and included Anoxybacillus,
Curtobacterium, and Yersinia. All three genera have pre-
viously been isolated from dairy products [45-48]. Anoxy-
bacillus is a thermophilic spore-former frequently isolated
from whole milk powder and nonfat dry milk and is some-
times used as a hygiene indicator in pasteurized diary
manufacture due to its high optimum growth temperature
[48]. Unpasteurized milk often contains the potential
pathogen Yersinia enterocolitica and the organism can be
found in curd samples when the milk is used to make
cheese. However, in one study, Y. enterocolitica has been
identified in one out of 265 pasteurized milk samples [47].
A total of 28 genera were identified only in the pasteur-
ized goat Gouda cheese in this study and included
Mannheimia, Leptotrichia, Balneimonas, Klebsiella, and
Pseudoalteromonas. Mannheimia and Leptotrichia may
have been part of the goat ecosystem which was transmit-
ted to the milk used in the manufacture of the cheeses.
The genus Mannheimia is comprised of bacteria respon-
sible for epizootic pneumonia and mastitis in goats, sheep,
and cattle [49], while Leptotrichia has been isolated from
goat foot lesions [50] and are normally found in the hu-
man oral cavity [51]. Balneimonas has not previously
been isolated from cheese, but has been isolated from
Suanzhou (Chinese fermented cereal gruel) samples
[52]. Bacteria in the genus Klebsiella, such as
Klebsiella pneumoniae, are human pathogens and
have also been found to cause spoilage in cheese via
gas production leading to early blowing of semi-hard
and hard cheeses [53]. Like many other organisms,
Klebsiella lack thermoresistance, which indicates con-
tamination of the cheeses most likely occurred during
or post-manufacture. Pseudoalteromonas are mesophi-
lic or psychrophilic marine bacteria that can survive
in environments with high salinity. The genera has
been identified in soft and semi-hard cow pasteurized
and unpasteurized cheeses as well as in cheese rinds
[8] and on the surfaces of smear-ripened cheeses [54,
55]. In one study, Pseudoalteromonas haloplanktis
comprised 17% of the total mapped reads of a
smear-ripened cheese as determined using 16S rDNA
metagenomics sequencing [54].
Overall, the majority of all the bacterial genera identi-
fied in this study were present in all three cheese regions
(under the rind, core, inside), however differences were
observed in population proportions. In the samples
taken under the rind, Staphylococcus and Tetragenococ-
cus were prevalent (9.6 and 4.8% of the total sequencing
reads for all cheeses, respectively). Large populations of
Staphylococcus on the surfaces of cheeses have been de-
tected previously [23, 56]. Tetragenococcus, a moderately
halophilic bacterial genus, has previously been detected
in unpasteurized hard cheeses and cheese rinds at 0.05
and 0.18%, respectively, but was not detected in soft or
semi-hard cheeses [8]. In addition, Brachybacterium,
Pseudoalteromonas, Yersinia, Klebsiella, and Weissella
were only detected in the under the rind samples.
Brachybacterium and Pseudoalteromonas are both halo-
philes, capable of growing in concentrations of salt as
high as 15-18% [57, 58]. Therefore, these organisms
may have contaminated the cheese during brining.
Yersinia and Klebsiella contain species which are poten-
tial human pathogens and are ubiquitous in the

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