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Advances in Microbiology, 2018, 8, 406-421

http://www.scirp.org/journal/aim

ISSN Online: 2165-3410

ISSN Print: 2165-3402

DOI:

10.4236/aim.2018.85027 May 31, 2018 406 Advances in Microbiology

Cultural Isolation and Characteristics of the

Blood Microbiome of Healthy Individuals

Stefan Panaiotov

, Georgi Filevski

, Michele Equestre

, Elena Nikolova

, Reni Kalfin

National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria

First Town’s Hospital, Patriarch Eftimii 37, Sofia, Bulgaria

Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy

Institute of Experimental Morphology, Pathology and Anthropology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Abstract

Background:

On the analogy of the non-pathogenic microbiota found in

oral

cavity, skin and gastrointestinal tract, existence of blood microbiota was co

firmed by DNA sequencing, but never deeply characterized. Hypothesis

for

the existence of dormant blood microbiota in healthy humans have been ar

sen and single species have been isolated. The aim of our study was

resuscitate and investigate the biodiversity of bacterial and fungal

dormant

blood microbiota in healthy individuals by blood culturing and NGS

DNA

sequencing.

Results:

Twenty eight blood samples of healthy individuals,

seven

for each blood type, were studied. Several culture media were tested.

Blood

microbiota resuscitation was performed in BHI broth supplemented with v

tamin K 1 mg/ml, 2% sucrose, 0.25% sodium citrate and 0.2% yeastolate

43˚C for 72 h. All tested blood samples were culture positive, as confirmed

Gram staining and TEM. TEM images demonstrated well defined cell stru

tures. Analysis for bacterial and eukaryotic species was performed by

16S

rRNA and ITS2 targeted sequencing. The obtained sequences were

clustered

(≥97% identity) in Operational Taxonomic Units (OTUs). Among

cultured

and uncultured samples we identified OTUs similarity with 47

bacterial

orders belonging to 15 phyla and 39 fungi orders blonging to 2 phyla. For

the

first time we demonstrated isolation and sequencing identification of

fungal

blood microbiota in healthy individuals. Blood-group differences

were

identified among the bacterial microbiome compositions.

Conclusion:

The

dormant blood microbiome is innate of the healthy individuals. Interventional

strategies to bind the host blood microbiome with the states of health

and

disease remain an unmet research goal.

Keywords

Blood Microbiota, Targeted Next Generation Sequencing, Operational

How to cite this paper:

Panaiotov, S.,

Filevski

, G., Equestre, M., Nikolova, E. and

Kalfin

, R. (2018)

Cultural Isolation and

Characteristics of the Blood Microbiome of

Healthy Individuals

Advances in Microb

ology

, 406-421.

https://doi.org/10.4236/aim.2018.85027

Received:

March 30, 2018

Accepted:

May 28, 2018

Published:

May 31, 2018

8 by authors and

Scientific

Research Publishing Inc.

This work is licensed under the Creative

Commons Attribution International

License (CC BY

4.0).

http://creativecommons.org/licenses/by/4.0/

Open Access

S. Panaiotov et al.

DOI:

10.4236/aim.2018.85027 407 Advances in Microbiology

Taxonomic Unit

1. Introduction

In the human and animal evolution many microbial species successfully adapted

to the macroorganism. Most of them could not be cultured and are proven indi-

rectly by DNA sequencing. On the bases of sampling 242 healthy adults at 18

different anatomical sites of the human body and sequencing the 16S rRNA

genes the presence of 5177 microbial taxonomic profiles was proven, but only

800 of them could be cultured [1]. The observation of cell-free DNAemia is well

described feature of the healthy blood [2] [3], but the presence of transient cul-

turable blood microbiota in the blood of healthy individuals could also be sup-

posed. We tested whether the presence of DNA in healthy blood is associated

with DNAemia or it is due to existing blood microbiota. It is proven that in the

blood of clinically healthy individuals microorganisms could persist for many

years without causing illness. The most investigated examples being the latent

tuberculosis.

Nevertheless the blood microbiome is still an enigma, its existence was proven

during the last 50 years. Indirect evidences for existence of bacteria residing in

erythrocytes have been predicted in the past by radiometric methods

[4]. In 1969

Tedeschi

et al

. reported on incorporation of nucleosides in human erythrocyte

attributed to the metabolic activity of mycoplasma or bacterial L-forms [4]. In

1977, Domingue and Schlegel identified in 7% of the blood specimens from

supposedly healthy individuals novel bacterial structures [5]. In 1993 the Bulga-

rian scientist Emil Kalfin experimentally proved by culturing and electron mi-

croscopy that microorganisms are multiplying in the erythrocytes of healthy

people [6]. Kalfin reported 100% positivity of the blood cultures. Subsequently

several other authors questioned the existence of the blood microbiome or

DNAemia in healthy individuals, Domingue (1977) [5], (1997) [7], Nikkari

et al.

(2001) [8], Mc Laughlin

et al

. (2002) [9], Moriyama

et al.

(2008) [10], Markova

(2015) [11], Damgaard (2015) [12] Dimova

et al.

(2017) [13], Gosiewski

et al.

(2017) [2] and Kowarsky

et al.

(2017) [3]. The authors reported supporting elec-

tron microscopy, cultural and molecular data in favour of the existence of the

blood microbiota in healthy individuals. Hypothesis on the bacterial structure of

the blood microbiota in healthy humans have been arisen [5] [6] [12], but

cultures for exhaustive microbiota analysis remain

a target. Rich bacterial

diversity in the blood of healthy individuals was confirmed by 16S rRNA genes

sequencing [14] and total RNA sequencing [15]. In 2016 Paise

et al.

demon-

strated that a diversified microbiome exists in healthy blood. Most of the blood

bacterial DNA was found located in the BC (93.74%), while RBCs contain more

bacterial DNA (6.23%) than the plasma (0.03%) [14]. A significant number of

bacterial species were also detected in the blood of healthy chickens [16] and cats

S. Panaiotov et al.

DOI:

10.4236/aim.2018.85027 408 Advances in Microbiology

by NGS analysis [17]. Resuscitation of dormant blood microbiota in healthy

individuals has been tested and single bacterial species have been isolated on

agar plates [12]. However, presence of eukaryotic microbial cells in the blood of

healthy individuals has never been challenged. Goal of our study was to

resuscitate the blood microbiota of healthy individuals and apply cultural, mi-

croscopic and NGS targeted sequencing methods for their characterization.

Here, we demonstrate that bacteria and fungi constitute a rich microbial diver-

sity in the blood of healthy humans by applying a successful culture resuscitation

strategy, DNA isolation and NGS targeted sequencing analysis. First preliminary

results of the study have been presented as poster at the ASM Microbe congress

2017 [18].

2. Materials and Methods

Ethical committee approval of the study (decision 38/14.07.2016) by the Institute

of Neurobiology, Bulgarian Academy of Sciences (BAS) and individual written

consent were obtained.

2.1. Culturing

Blood of 28 healthy volunteers was collected in Vacutainer tubes with K

EDTA

as anticoagulant (Vacutainer K3E, BD, USA), 7 samples per blood group. The

blood samples were divided in two parts. One part for culturing (3 mL) and

another part for direct DNA isolation (7 mL). All blood samples were tested for

sterility by growing on Sabouraud and blood agar.

We applied a modified resuscitation strategy previously developed by Emil

Kalfin [6]. Three ml of blood sample were added to 22 ml of culture medium.

Culturing was performed in sterile 50 ml polypropylene Falcon tubes (Corning

Inc, USA). The culture base medium was composed by Brain Hearth Infusion

(BHI, Difco, USA) medium and 0.2% yeastolate (Difco) adjusted at pH 6.8 and

sterilized. Sterile (D+) sucrose at 10% final concentration and water-soluble

form of vitamine K

- menadione sodium bisulfite (Sigma-Aldrich, USA) in

concentration of 1 mg/ml sterilized by filtration were added to the base medium.

Resuscitation growth was induced at 43˚C at 24 h, but 72 h was found to be op-

timal time for growing blood microbiota in liquid cultures. The agar medium for

subculturing contained 1.2% Noble agar (Difco, BD, USA). Isolated blood mi-

crobiota was confirmed by Gram staining and 16S rRNA genes PCR analysis.

Gram staining was directly applied on the cultured sample.

2.2. Transmission Electron Microscopy (TEM)

Blood samples for TEM were processed within 1 hour after collection. Sterile

heparinized blood collected in Vacutainer tube (BD, USA) was diluted 1:3 with

PBS pH 7.4 (Thermo Fisher Scientific, USA) and centrifuged at 400 g for 30 min

on Histopaque-1077 (Sigma-Aldrich, USA) density gradient. The peripheral

blood mononuclear cells were removed from the gradient, washed 3 times in

S. Panaiotov et al.

DOI:

10.4236/aim.2018.85027 409 Advances in Microbiology

PBS, then washed in RPMI 1640 (Sigma-Aldrich, USA) medium with 10% FCS

(Sigma-Aldrich, USA) and processed for TEM. The cells were resuspended in

3% low gelling temperature agarose (Sigma-Aldrich, USA) and put on ice. The

solidified agarose was cut into 1 mm

cubes, then fixed in 2.5% glutaraldehyde

(Sigma-Aldrichm USA), postfixed in 1% osmium tetraoxide (Sigma-Aldrich,

USA) and dehydrated in increasing concentrations of 30, 50, 70, 96% of ethanol

for 10 min each, 100% ethanol for 2 × 20 min and propylene oxide for 2 × 20

min. Specimens were impregnated in propylene oxide: Durcupan ACM (Sig-

ma-Aldrich, USA) 1:1 and embedded in Durcupan ACM. Polymerisation was

carried out at 60°C for 18 h in an oven. The samples were thin cut to 20 30 nm

and observed under transmission electron microscope Opton EM 109 (Zeiss,

Germany).

2.3. Physico-Chemical Analysis and Radioresistance

Disruption of cells with 0.1 mm glass beads with beat beater machine

(Mini-beatbeater, Biospec Products, USA), treatment in microwave oven at 850

W and disintegration with ultrasound (MSE, UK) on ice was applyed. Integrity

of the cells in fresh cultures was tested by treatments

with 4 M guanidine thioci-

anate, 10% NaOH, 10% KOH, 10% CH

COOH, 10% HCl and 10% H

for 30

min at 37˚C.

To test the level of the ionizing radiation that the microbiota are able to

withstand we applied gamma irradiation with

Co (Isledovatel MPX-

-25M,

TENEX, Moscow, RU) at 15, 20 and 25 kGy to 5 ml of blood from three healthy

donors and erythrocyte concentrate of blood group A, Rh(+). The erythrocyte

concentrate was a sample produced by collecting blood from several blood do-

nors at the National Center for Transfusiology and Hematology in Sofia, Bulga-

ria. In order to control the quality of irradiation two control blood samples were

spiked with

Candida guiliermondii

and

Candida albicans

with 10

cells. After ir-

radiation with dose of 15 kGy, fungi inoculated control samples were tested for

growth on Sabouraud agar. Control plates were negative after two weeks of cul-

turing at 30˚C.

2.4. DNA Isolation and Sequencing Analysis

Seven commercial kits were tested for DNA isolation of the cultured blood mi-

crobiota: Tissue and cells genomic Prep kit and Blood genomic Prep kit, (GE,

USA); Genomic DNA purification kit (Thermo

Fisher Scientific, USA); QIAamp

DNA isolation kit (Qiagen, Germany); NucleoSpin Soil and NucleoSpin DNA

Stool kit (Macherey-Nagel, Germany); and RIBO-prep kit (Ecoli s.r.o., Slovakia).

Most of the DNA extraction kits: Tissue and cells genomic Prep kit and Blood

genomic Prep kit, (GE, USA); Genomic DNA purification kit, (Thermo Fisher

Scientific, USA); QIAamp DNA isolation kit, (Qiagen, Germany) and the stan-

dard CTAB phenol/chlorophorm DNA extraction protocol were not successful

for DNA extraction of the cultured blood microbiota. Successful DNA extrac-

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