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"Iida, Tetsuya"
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Metagenomic Analysis Reveals Dynamic Changes of Whole Gut Microbiota in the Acute Phase of Intensive Care Unit Patients
Background
Metagenomic analysis targeting the 16S rRNA gene has made it possible to characterize the vast array of microorganisms contained in the gut.
Aim
The purpose of this study was to evaluate how gut microbiota change in intensive care unit (ICU) patients in the acute phase after admission.
Methods
This prospective observational cohort study investigated 12 patients admitted to a single ICU of a large urban tertiary referral hospital. All patients were mechanically ventilated on admission. Fecal samples were collected from patients on days 1–2, 2–4, 5–8, and 7–10 after admission. DNA was extracted from fecal samples, and 16S rRNA deep sequencing was performed to monitor gut changes.
Results
Bacteria belonging to the phyla
Firmicutes
or
Bacteroidetes
were predominant in each sample. We observed serial dynamic changes in the percentages of
Bacteroidetes
and
Firmicutes
that were significantly altered during study period (
p
< 0.05). A ratio of
Bacteroidetes
to
Firmicutes
(B/F ratio) of >10 was seen in four of the six patients who died, whereas a B/F ratio of <0.10 was seen in only one of the six deaths. None of the survivors had a B/F ratio of >10 or <0.10. There was a statistical difference in the B/F ratio between the dead patients and survivors (
p
= 0.022).
Conclusions
Dynamic changes in gut microbiota at the phylum level of ICU patients during the acute phase were identified by high-throughput DNA sequencing. An extreme imbalance in gut microbiota may be associated with prognosis in critically ill patients.
Journal Article
Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation
Background
Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN.
Methods
Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using
Ahr
-deficient mice.
Results
Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (
Il
)-
10
and
Il-22
in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4
+
T cells and IL-22-producing CD3
−
RORγt
+
cells, but not CD4
+
Foxp3
+
regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in
Ahr
-deficient mice, in association with diminished regulatory cytokine production.
Conclusions
IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.
Journal Article
Impact of sleep on the microbiome of oral biofilms
Dysbiosis of the oral microbiome is associated with diseases such as periodontitis and dental caries. Because the bacterial counts in saliva increase markedly during sleep, it is broadly accepted that the mouth should be cleaned before sleep to help prevent these diseases. However, this practice does not consider oral biofilms, including the dental biofilm. This study aimed to investigate sleep-related changes in the microbiome of oral biofilms by using 16S rRNA gene sequence analysis. Two experimental schedules—post-sleep and pre-sleep biofilm collection—were applied to 10 healthy subjects. Subjects had their teeth and oral mucosa professionally cleaned 7 days and 24 h before sample collection. Samples were collected from several locations in the oral cavity: the buccal mucosa, hard palate, tongue dorsum, gingival mucosa, tooth surface, and saliva. Prevotella and Corynebacterium had higher relative abundance on awakening than before sleep in all locations of the oral cavity, whereas fluctuations in Rothia levels differed depending on location. The microbiome in different locations in the oral cavity is affected by sleep, and changes in the microbiome composition depend on characteristics of the surfaces on which oral biofilms form.
Journal Article
Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia
Lypd8 protein derived from intestinal epithelial cells binds to flagellated bacteria to reduce their motility, which limits the entry of Gram-negative bacteria into the inner colonic mucus and prevents invasion of colonic epithelia.
Lypd8 separates microbiota from epithelia
This paper shows that the intestinal epithelial cell derived protein Lypd8, a member of member of the Ly6/PLAUR superfamily, binds to flagellated bacteria. In doing so it reduces the bacteria's motility, limits the entry of Gram-negative bacteria into the inner colonic mucus, and prevents invasion into colonic epithelium.
Colonic epithelial cells are covered by thick inner and outer mucus layers
1
,
2
. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis
3
,
4
,
5
,
6
. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified
7
,
8
,
9
,
10
,
11
. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including
Proteus mirabilis
. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia.
Lypd8
−/−
mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in
Lypd8
−/−
mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.
Journal Article
Genomic analysis of carbapenem- and colistin-resistant Klebsiella pneumoniae complex harbouring mcr-8 and mcr-9 from individuals in Thailand
The surge in mobile colistin-resistant genes (
mcr
) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring
mcr
in 2015–2020. In this study, we aimed to examine the genomic characteristics and phonotypes of
mcr-8
and
mcr-9
harbouring carbapenem-resistant
K. pneumoniae
complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as
K. pneumoniae
(CRKP) ST147 and strain AMR20200784 as
K. quasipneumoniae
(CRKQ) ST476, harbouring
mcr-8
and
mcr-9
, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (
pmrB, pmrC, ramA,
and
lpxM
), while CRKP showed two substitutions in
crrB
,
pmrB, pmrC, lpxM
and
lapB
. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for
mcr-8
-carrying CRKP isolate and 32 µg/ml for
mcr-9
-carrying CRKQ isolate. In addition, CRKP harbouring
mcr-8
carried
bla
NDM
, while CRKQ harbouring
mcr-9
carried
bla
IMP
, conferring carbapenem resistance. Analysis of plasmid replicon types carrying
mcr-8
and
mcr-9
showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried
bla
IMP-14
on an IncC plasmid, while the CRKP harboured
bla
NDM-1
on an FIB plasmid. This finding provides a comprehensive insight into another
mcr
-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were
bla
CTX-M-15
,
bla
SHV-11
,
bla
OXA-1
,
aac(6′)-Ib-cr, aph(3′)-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA,
and
qacE
, while those detected in CRKQ were
bla
OKP-B-15
,
qnrA1, oqxA, oqxB, sul1, fosA,
and
qacE
. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent
mcr
-harbouring CRE and the need for rational drug use in all sectors.
Journal Article
The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus
Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V . parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB . VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V . parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo , indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V . parahaemolyticus .
Journal Article
Vibrio parahaemolyticus VtrA is a membrane-bound regulator and is activated via oligomerization
Vibrio parahaemolyticus is a Gram-negative pathogen that causes food-borne gastroenteritis. A major virulence determinant of the organism is a type III secretion system (T3SS2) encoded on a pathogenicity island, Vp-PAI. Vp-PAI gene expression is regulated by two transcriptional regulators, VtrA and VtrB, whose N-terminal regions share homology with an OmpR-family DNA-binding domain. VtrA activates the gene expression of VtrB, which in turn activates Vp-PAI gene expression; however, the mechanism of this transcriptional activation by VtrA is not well understood. In this study, we determined that VtrA is a membrane protein with a transmembrane (TM) domain, which was required for its transcriptional regulatory activity. Although the N-terminal region of VtrA alone is insufficient for its transcriptional regulatory activity, forced oligomerization using the leucine-zipper dimerization domain of yeast GCN4 conferred transcriptional regulatory activity and a greater affinity for the promoter region of vtrB. A ToxR-based assay demonstrated that VtrA oligomerizes in vivo. We also showed that bile, a host-derived activator of VtrA, induces the oligomerization of VtrA, which requires the C-terminal domain. The promoter region of vtrB contained repetitive T-rich DNA elements, which are important for vtrB transcriptional activation and are conserved among T3SS2-possessing Vibrio species. These findings propose that VtrA is active as oligomers, which may facilitate its N-terminus binding the target DNA, thus enhancing its transcriptional regulatory activity.
Journal Article
Comprehensive subspecies identification of 175 nontuberculous mycobacteria species based on 7547 genomic profiles
The prevalence of nontuberculous mycobacteria (NTM) pulmonary diseases has been increasing worldwide. NTM consist of approximately 200 species and distinguishing between them at the subspecies level is critical to treatment. In this study, we sequenced 63 NTM genomes, 27 of which were newly determined, by hybrid assembly using sequencers from Illumina and Oxford Nanopore Technologies (ONT). This analysis expanded the available genomic data to 175 NTM species and redefined their subgenus classification. We also developed a novel multi-locus sequence typing (MLST) database based on 184 genes from 7547 assemblies and an identification software, mlstverse, which can also be used for detecting other bacteria given a suitable MLST database. This method showed the highest sensitivity and specificity amongst conventional methods and demonstrated the capacity for rapid detection of NTM, 10 min of sequencing of the ONT MinION being sufficient. Application of this methodology could improve disease epidemiology and increase the cure rates of NTM diseases.
Journal Article
Impacts of sleep on the characteristics of dental biofilm
Dental biofilm present on the tooth surface is associated with oral diseases, such as dental caries and periodontal disease. Because bacterial numbers rapidly increase in saliva during sleep, oral care before sleeping is recommended for the prevention of chronic oral diseases. However, temporal circadian changes in the quantity and quality of dental biofilms are poorly understood. This study aimed to investigate the impacts of sleeping on dental biofilm amounts and compositions by using an in situ model. The use of this in situ model enabled us to investigate dental biofilm formed in the oral cavity and to perform a quantitative analysis. Subjects began wearing oral splints in the morning or before sleeping, and biofilm samples were collected at 8, 16, and 24 h after the subjects began wearing oral splints; these samples were then used in various experiments. No significant changes in the numbers of biofilm-forming bacteria were caused by sleep. However, the relative abundances of genera related to periodontitis (i.e.,
Fusobacterium
and
Prevotella
) increased after awakening. In conclusion, the numbers of biofilm-forming bacteria were not affected by sleep, and the abundances of obligate anaerobes increased after sleep. This research may aid in defining efficacious preventive oral care.
Journal Article