21. Food Microbiol. 2015 Apr;46:342-56. doi: 10.1016/j.fm.2014.08.023. Epub 2014 Sep
Połka J(1), Rebecchi A(2), Pisacane V(2), Morelli L(1), Puglisi E(3).
(1)Istituto di Microbiologia, Facoltà di Scienze Agrarie, Alimentari ed
Ambientali, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122
Piacenza, Italy. (2)Centro Ricerche Biotecnologiche, Università Cattolica del
Sacro Cuore, Via Milano 24, 26100 Cremona, Italy. (3)Istituto di Microbiologia,
Facoltà di Scienze Agrarie, Alimentari ed Ambientali, Università Cattolica del
Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy. Electronic address:
Food Microbiol. 2015 Jun;48:191.
The bacterial diversity involved in food fermentations is one of the most
important factors shaping the final characteristics of traditional foods.
Knowledge about this diversity can be greatly improved by the application of
high-throughput sequencing technologies (HTS) coupled to the PCR amplification of
the 16S rRNA subunit. Here we investigated the bacterial diversity in batches of
Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage
that is typical of a regional area of Northern Italy. Salami samples from 6
different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw
meat at time 0 and casing samples at 21 days of ripening where also analysed, and
the effect of starter addition was included in the experimental set-up.
Culture-based microbiological analyses and PCR-DGGE were carried out in order to
be compared with HTS results. A total of 722,196 high quality sequences were
obtained after trimming, paired-reads assembly and quality screening of raw reads
obtained by Illumina MiSeq sequencing of the two bacterial 16S hypervariable
regions V3 and V4; manual curation of 16S database allowed a correct taxonomical
classification at the species for 99.5% of these reads. Results confirmed the
presence of main bacterial species involved in the fermentation of salami as
assessed by PCR-DGGE, but with a greater extent of resolution and quantitative
assessments that are not possible by the mere analyses of gel banding patterns.
Thirty-two different Staphylococcus and 33 Lactobacillus species where identified
in the salami from different producers, while the whole data set obtained
accounted for 13 main families and 98 rare ones, 23 of which were present in at
least 10% of the investigated samples, with casings being the major sources of
the observed diversity. Multivariate analyses also showed that batches from 6
local producers tend to cluster altogether after 21 days of ripening, thus
indicating that HTS has the potential for fine scale differentiation of local
Copyright © 2014 Elsevier Ltd. All rights reserved.
PMID: 25475305 [PubMed - indexed for MEDLINE]
22. BMC Genomics. 2016 Jan 14;17:55. doi: 10.1186/s12864-015-2194-9.
D'Amore R(1), Ijaz UZ(2), Schirmer M(3), Kenny JG(4), Gregory R(5), Darby AC(6),
Shakya M(7), Podar M(8), Quince C(9), Hall N(10).
(1)Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB,
UK. email@example.com. (2)School of Engineering, University of
Glasgow, Glasgow, G12 8LT, UK. Umer.Ijaz@glasgow.ac.uk. (3)School of Engineering,
University of Glasgow, Glasgow, G12 8LT, UK. firstname.lastname@example.org.
(4)Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB,
UK. email@example.com. (5)Institute of Integrative Biology, University of
Liverpool, Liverpool, L69 7ZB, UK. firstname.lastname@example.org. (6)Institute of Integrative
Biology, University of Liverpool, Liverpool, L69 7ZB, UK. (7)Department of
Biological Sciences, Dartmouth College, Hanover, NH03755, USA.
email@example.com. (8)Biosciences Division, Oak Ridge National Laboratory,
Oak Ridge, 37831, TN, USA. firstname.lastname@example.org. (9)Warwick Medical School, University
of Warwick, Warwick, CV4 7AL, UK. C.Quince@warwick.ac.uk. (10)Institute of
Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
BACKGROUND: In the last 5 years, the rapid pace of innovations and improvements
in sequencing technologies has completely changed the landscape of metagenomic
and metagenetic experiments. Therefore, it is critical to benchmark the various
methodologies for interrogating the composition of microbial communities, so that
we can assess their strengths and limitations. The most common phylogenetic
marker for microbial community diversity studies is the 16S ribosomal RNA gene
and in the last 10 years the field has moved from sequencing a small number of
amplicons and samples to more complex studies where thousands of samples and
multiple different gene regions are interrogated.
RESULTS: We assembled 2 synthetic communities with an even (EM) and uneven (UM)
distribution of archaeal and bacterial strains and species, as metagenomic
control material, to assess performance of different experimental strategies. The
2 synthetic communities were used in this study, to highlight the limitations and
the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific
Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We
describe an extensive survey based on synthetic communities using 3 experimental
designs (fusion primers, universal tailed tag, ligated adaptors) across the 9
hypervariable 16S rDNA regions. We demonstrate that library preparation
methodology can affect data interpretation due to different error and chimera
rates generated during the procedure. The observed community composition was
always biased, to a degree that depended on the platform, sequenced region and
primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing
is still quantitative, in that relative changes in abundance of taxa between
samples can be recovered, despite these biases.
CONCLUSION: We have assessed a range of experimental conditions across several
next generation sequencing platforms using the most up-to-date configurations. We
propose that the choice of sequencing platform and experimental design needs to
be taken into consideration in the early stage of a project by running a small
trial consisting of several hypervariable regions to quantify the discriminatory
power of each region. We also suggest that the use of a synthetic community as a
positive control would be beneficial to identify the potential biases and
procedural drawbacks that may lead to data misinterpretation. The results of this
study will serve as a guideline for making decisions on which experimental
condition and sequencing platform to consider to achieve the best microbial
PMID: 26763898 [PubMed - indexed for MEDLINE]
23. BMC Microbiol. 2016 Jul 11;16(1):145. doi: 10.1186/s12866-016-0758-8.
Mwaikono KS(1,)(2), Maina S(3), Sebastian A(4), Schilling M(5), Kapur V(6,)(5),
(1)Department of Science and Laboratory Technology, Dar es Salaam Institute of
Technology, Dar es Salaam, Tanzania. email@example.com. (2)School of Life
Sciences and Bioengineering, The Nelson Mandela African Institution of Science
and Technology, P.O. Box 447, Arusha, Tanzania. firstname.lastname@example.org.
(3)BecA-ILRI Hub International Livestock Research Institute, P. O. Box 30709,
Nairobi, Kenya. (4)Departments of Biochemistry and Molecular Biology, W238A
Millennium Science Complex, Penn State University, University Park, PA, 16802,
USA. (5)Huck Institutes of Life Sciences, Molecular Cellular and Integrative
Biosciences, the Pennsylvania State University, 204 Wartik Laboratories,
University Park, PA, 16802, USA. (6)School of Life Sciences and Bioengineering,
The Nelson Mandela African Institution of Science and Technology, P.O. Box 447,
Arusha, Tanzania. (7)Genome Sciences Centre, Faculty of Veterinary Medicine,
Sokoine University of Agriculture, Morogoro, Tanzania.
BACKGROUND: Multiple types of solid waste in developing countries is disposed of
together in dumpsites where there is interaction between humans, animals and the
bacteria in the waste. To study the bacteria at the dumpsite and the associated
risks, previous studies have focused on culturable, leaving behind a great number
of unculturable bacteria. This study focuses on a more comprehensive approach to
study bacteria at the dumpsite. Since the site comprised of unsorted wastes, a
qualitative survey was first performed to identify the variety of solid waste as
this has influence on the microbial composition. Thus, domestic (Dom), biomedical
(Biom), river sludge (Riv), and fecal material of pigs scavenging on the dumpsite
(FecD) were sampled. Total DNA was extracted from 78 samples and the v4-16S rRNA
amplicons was characterized using an Illumina MiSeq platform.
RESULTS: A total of 8,469,294 sequences passed quality control. Catchall analysis
predicted a mean of 8243 species per sample. Diversity was high with an average
InvSimpson index of 44.21 ± 1.44. A total of 35 phyla were detected and the
predominant were Firmicutes (38 %), Proteobacteria (35 %), Bacteroidetes (13 %)
and Actinobacteria (3 %). Overall 76,862 OTUs were detected, however, only 20 %
were found more than 10 times. The predominant OTUs were Acinetobacter (12.1 %),
Clostridium sensu stricto (4.8 %), Proteinclasticum and Lactobacillus both at
(3.4 %), Enterococcus (2.9 %) and Escherichia/Shigella (1.7 %). Indicator
analysis (P ≤ 0.05, indicator value ≥ 70) shows that Halomonas, Idiomarina,
Tisierella and Proteiniclasticum were associated with Biom; Enterococcus,
Bifidobacteria, and Clostridium sensu stricto with FecD and Flavobacteria,
Lysobacter and Commamonas to Riv. Acinetobacter and Clostridium sensu stricto
were found in 62 % and 49 % of all samples, respectively, at the relative
abundance of 1 %. None of OTUs was found across all samples.
CONCLUSIONS: This study provides a comprehensive report on the abundance and
diversity bacteria in municipal dumpsite. The species richness reported here
shows the complexity of this man-made ecosystem and calls for further research to
assess for a link between human diseases and the dumpsite. This would provide
insight into proper disposal of the waste, as well as, limit the risks to human
health associated with the dumpsite.
PMID: 27400733 [PubMed - in process]
24. Insect Sci. 2015 Oct;22(5):606-18. doi: 10.1111/1744-7917.12155. Epub 2014 Nov
Saraithong P(1,)(2), Li Y(2), Saenphet K(1), Chen Z(2), Chantawannakul P(1,)(3).
(1)Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai,
50200, Thailand. (2)Department of Basic Science and Craniofacial Biology, New
York University College of Dentistry, New York, 10010, USA. (3)Materials Science
Research Center, Faculty of Science, Chiang Mai University, Chiang Mai, 50200,
This study characterizes the colonization and composition of bacterial flora in
dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and
distribution among different sampling locations. A. florea larvae were collected
from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted
from each larva using the phenol-chloroform method. Denaturing gradient gel
electrophoresis was performed, and the dominant bands were excised from the gels,
cloned, and sequenced for bacterial species identification. The result revealed
similarities of bacterial community profiles in each individual colony, but
differences between colonies from the same and different locations. A. florea
larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5
classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli,
and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus,
Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from
uncultured bacterial species. The classes with the highest abundance of bacteria
were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%),
Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly,
uncultured bacterial species were identified (12%). Environmental bacterial
species, such as Saccharibacter floricola, were also found. This is the first
study in which sequences closely related to Melissococcus plutonius, the causal
pathogen responsible for European foulbrood, have been identified in Thai A.
© 2014 Institute of Zoology, Chinese Academy of Sciences.
PMID: 25393530 [PubMed - indexed for MEDLINE]
25. Front Microbiol. 2016 Aug 23;7:1297. doi: 10.3389/fmicb.2016.01297. eCollection
Fischer MA(1), Güllert S(2), Neulinger SC(3), Streit WR(2), Schmitz RA(1).
(1)Department of Biology, Institute for General Microbiology,
Christian-Albrechts-Universität zu Kiel Kiel, Germany. (2)Biozentrum Klein
Flottbek, Institute of Microbiology & Biotechnology, Universität Hamburg Hamburg,
Germany. (3)Department of Biology, Institute for General Microbiology,
Christian-Albrechts-Universität zu KielKiel, Germany; omics2view.consulting
The application of next-generation sequencing technology in microbial community
analysis increased our knowledge and understanding of the complexity and
diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal
domain was often not particularly addressed in the analysis of microbial
communities. Consequently, established primers specifically amplifying the
archaeal 16S ribosomal gene region are scarce compared to the variety of primers
targeting bacterial sequences. In this study, we aimed to validate archaeal
primers suitable for high throughput next generation sequencing. Three archaeal
16S primer pairs as well as two bacterial and one general microbial 16S primer
pairs were comprehensively tested by in-silico evaluation and performing an
experimental analysis of a complex microbial community of a biogas reactor. The
results obtained clearly demonstrate that comparability of community profiles
established using different primer pairs is difficult. 16S rRNA gene data derived
from a shotgun metagenome of the same reactor sample added an additional
perspective on the community structure. Furthermore, in-silico evaluation of
primers, especially those for amplification of archaeal 16S rRNA gene regions,
does not necessarily reflect the results obtained in experimental approaches. In
the latter, archaeal primer pair ArchV34 showed the highest similarity to the
archaeal community structure compared to observed by the metagenomic approach and
thus appears to be the appropriate for analyzing archaeal communities in biogas
reactors. However, a disadvantage of this primer pair was its low specificity for
the archaeal domain in the experimental application leading to high amounts of
bacterial sequences within the dataset. Overall our results indicate a rather
limited comparability between community structures investigated and determined
using different primer pairs as well as between metagenome and 16S rRNA gene
amplicon based community structure analysis. This finding, previously shown for
Bacteria, was as well observed for the archaeal domain.
PMID: 27602022 [PubMed]