, and
are phenotypically and genotypically closely related, and together comprise the
group. Although the strains of this group are commercially valuable as probiotics, the taxonomic status and ...nomenclature of the
group have long been contentious because of the difficulties in identifying these three species by using the most frequently used genotypic methodology of 16S rRNA gene sequencing. Long used as the gold standard for species classification, DNA-DNA hybridization is laborious, requires expert skills, and is difficult to use routinely in laboratories. Currently, genome-based comparisons, including average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), are commonly applied to bacterial taxonomy as alternatives to the gold standard method for the demarcating phylogenetic relationships. To establish quick and accurate methods for identifying strains in the
group at the species and subspecies levels, we developed species- and subspecies-specific identification methods based on housekeeping gene sequences and whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral pattern analysis. By phylogenetic analysis based on concatenated housekeeping gene sequences (
, and
), 53 strains were separated into four clusters corresponding to the four species:
and
, and
sp. nov. A multiplex minisequencing assay using single nucleotide polymorphism (SNP)-specific primers based on the
gene sequences and species-specific primers based on the
gene sequences provided high resolution that enabled the strains at the species level to be identified as
, and
. By MALDI-TOF MS analysis coupled with an internal database and ClinProTools software, species- and subspecies-level
group strains were identified based on reliable scores and species- and subspecies-specific MS peaks. The
strains were distinguished clearly at the subspecies level based on subspecies-specific MS peaks. This article describes the rapid and accurate methods used for identification and classification of strains in the
group based on housekeeping gene sequences and MALDI-TOF MS analysis as well as the novel speciation of this group including
.
sp. nov. and '
' by genome-based methods.
Chronic obstructive pulmonary disease (COPD) is a progressive, life-threatening lung disease with increasing prevalence and incidence worldwide. Increasing evidence suggests that lung microbiomes ...might play a physiological role in acute exacerbations of COPD. The objective of this study was to characterize the association of the microbiota and exacerbation risk or airflow limitation in stable COPD patients.
The sputum microbiota from 78 COPD outpatients during periods of clinical stability was investigated using 16S rRNA V3-V4 amplicon sequencing. The microbiome profiles were compared between patients with different risks of exacerbation, i.e., the low risk exacerbator (LRE) or high risk exacerbator (HRE) groups, and with different airflow limitation severity, i.e., mild to moderate (FEV1 ≥ 50; PFT I) or severe to very severe (FEV1 < 50; PFT II).
The bacterial diversity (Chao1 and observed OTUs) was significantly decreased in the HRE group compared to that in the LRE group. The top 3 dominant phyla in sputum were Firmicutes, Actinobacteria, and Proteobacteria, which were similar in the HRE and LRE groups. At the genus level, compared to that in the LRE group (41.24%), the proportion of Streptococcus was slightly decreased in the HRE group (28.68%) (p = 0.007). However, the bacterial diversity and the proportion of dominant bacteria at the phylum and genus levels were similar between the PFT I and PFT II groups. Furthermore, the relative abundances of Gemella morbillorum, Prevotella histicola, and Streptococcus gordonii were decreased in the HRE group compared to those in the LRE group according to linear discriminant analysis effect size (LEfSe). Microbiome network analysis suggested altered bacterial cooperative regulation in different exacerbation phenotypes. The proportions of Proteobacteria and Neisseria were negatively correlated with the FEV1/FVC value. According to functional prediction of sputum bacterial communities through Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis, genes involved in lipopolysaccharide biosynthesis and energy metabolism were enriched in the HRE group.
The present study revealed that the sputum microbiome changed in COPD patients with different risks of exacerbation. Additionally, the bacterial cooperative networks were altered in the HRE patients and may contribute to disease exacerbation. Our results provide evidence that sputum microbiome community dysbiosis is associated with different COPD phenotypes, and we hope that by understanding the lung microbiome, a potentially modifiable clinical factor, further targets for improved COPD therapies during the clinically stable state may be elucidated.
Attention-deficit/hyperactivity disorder (ADHD) is a common childhood mental disorder with undetermined pathophysiological mechanisms. The gut microbiota and immunological dysfunction may influence ...brain functions and social behaviours. In the current study, we aimed to explore the correlation of gut microbiome imbalance and inflammation in the pathophysiology of ADHD. Forty-one children with ADHD and thirty-nine healthy-control (HC) individuals were recruited. Faecal samples from all participants were collected and submitted for 16 S rRNA V3-V4 amplicon microbiome sequencing analysis. The plasma levels of 10 cytokines, including TNF-α, IL-6, IL-1β, IL-2, IL-10, IL-13, IL-17A, IFN-α2, IFN-γ, and MCP-1, were determined using a custom-made sandwich enzyme-linked immunosorbent assay (ELISA) developed by Luminex Flowmetrix. There was no significant difference between the ADHD and HC groups in species diversity in the faeces, as determined with α-diversity and β-diversity analysis. In the ADHD group, three differentially abundant taxonomic clades at the genus level were observed, namely Agathobacter, Anaerostipes, and Lachnospiraceae. Top differentially abundant bacteria and representative biological pathways were identified in children with ADHD using linear discriminant analysis (LDA) effect size (LEfSe), and the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis, respectively. The plasma levels of TNF-α were significantly lower in children with ADHD than in HCs. Within the ADHD group, the levels of TNF-α were negatively correlated with ADHD symptoms and diversity of the gut microbiome. Our study provides new insights into the association between gut microbiome dysbiosis and immune dysregulation, which may contribute to the pathophysiology of ADHD.
Genomic changes in Mycoplasma pneumoniae caused by adaptation to environmental or ecologic pressures are poorly understood. We collected M. pneumoniae from children who had confirmed pneumonia in ...Taiwan during 2017-2020. We used whole-genome sequencing to compare these isolates with a worldwide collection of current and historical clinical strains for characterizing population structures. A phylogenetic tree for 284 strains showed that all sequenced strains consisted of 5 clades: T1-1 (sequence type ST1), T1-2 (mainly ST3), T1-3 (ST17), T2-1 (mainly ST2), and T2-2 (mainly ST14). We identified a putative recombination block containing 6 genes (MPN366‒371). Macrolide resistance involving 23S rRNA mutations was detected for each clade. Clonal expansion of macrolide resistance occurred mostly within subtype 1 strains, of which clade T1-2 showed the highest recombination rate and genome diversity. Functional characterization of recombined regions provided clarification of the biologic role of these recombination events in the evolution of M. pneumoniae.
Incidence of invasive pneumococcal disease caused by antimicrobial-resistant Streptococcus pneumoniae types not included in pneumococcal conjugate vaccines has increased, including a penicillin- and ...meropenem-resistant serotype 15A-ST63 clone in Japan. During 2013–2017, we collected 206 invasive pneumococcal isolates in Taiwan for penicillin and meropenem susceptibility testing. We found serotypes 15B/C-ST83 and 15A-ST63 were the most prevalent penicillin- and meropenem-resistant clones. A transformation study confirmed that penicillin-binding protein (PBP) 2b was the primary meropenem resistance determinant, and PBP1a was essential for high-level resistance. The rate of serotype 15B/C-ST83 increased during the study. All 15B/C-ST83 isolates showed an ermB macrolide resistance genotype. Prediction analysis of recombination sites revealed 12 recombination regions in 15B/C-ST83 compared with the S. pneumoniae Spain23F-ST81 genome. Pneumococcal clones rapidly recombine to acquire survival advantages and undergo local expansion under the selective pressure exerted by vaccines and antimicrobial drugs. The spread of 15B/C-ST83 is alarming for countries with high antimicrobial pressure.
Human breast milk is widely recognized as the best source of nutrients for healthy growth and development of infants; it contains a diverse microbiota. Here, we characterized the diversity of the ...microbiota in the breast milk of East Asian women and assessed whether delivery mode influenced the microbiota in the milk of healthy breast-feeding mothers. We profiled the microbiota in breast milk samples collected from 133 healthy mothers in Taiwan and in six regions of mainland China (Central, East, North, Northeast, South, and Southwest China) by using 16S rRNA pyrosequencing. Lactation stage (months postpartum when the milk sample was collected) and maternal body mass index did not influence the breast milk microbiota. Bacterial composition at the family level differed significantly among samples from the seven geographical regions. The five most predominant bacterial families were
(mean relative abundance: 24.4%),
(14.0%),
(12.2%),
(6.2%), and
(4.8%). The microbial profiles were classified into three clusters, driven by
(abundance in Cluster 1: 42.1%),
(Cluster 2: 48.5%), or
(Cluster 3: 26.5%). Microbial network analysis at the genus level revealed that the abundances of the Gram-positive
,
, and
were negatively correlated with those of the Gram-negative
,
,
,
, and
. Milk from mothers who had undergone Caesarian section (C-section group) had a significantly higher abundance of
(
< 0.05) and a higher number of unique unclassified operational taxonomic units (OTUs) (
< 0.001) than that from mothers who had undergone vaginal delivery (vaginal group). These findings revealed that (i) geographic differences in the microbial profiles were found in breast milk from mothers living in Taiwan and mainland China, (ii) the predominant bacterial families
,
, and
were key components for forming three respective clusters, and (iii) a significantly greater number of unique OTUs was found in the breast milk from mothers who had undergone C-section than from those who had delivered vaginally.
Background: Probiotics have been previously reported to reduce the incidence of necrotizing enterocolitis (NEC) in extremely preterm infants, but the mechanisms by which the probiotics work remain ...unknown. We aimed to investigate the effects of probiotics on the gut microbiota of extremely preterm infants. Methods: A prospective cohort study was conducted on 120 extremely preterm neonates (gestational age ≤ 28 weeks) between August 2019 and December 2021. All neonates were divided into the study (receiving probiotics) and the control (no probiotics) groups. Multivariate logistic regression analysis was performed to investigate the significantly different compositions of gut microbiota between these two groups. The effects of probiotics on the occurrence of NEC and late-onset sepsis were also investigated. Results: An increased abundance of Lactobacillus was noted in neonates who received the probiotics (AOR 4.33; 95% CI, 1.89–9.96, p = 0.009) when compared with the control group. Subjects in the probiotic group had significantly fewer days of total parenteral nutrition (median interquartile range, IQR) 29.0 (26.8–35.0) versus 35.5 (27.8–45.0), p = 0.004) than those in the control group. The probiotic group had a significantly lower rate of late-onset sepsis than the control group (47.1% versus 70.0%, p = 0.015), but the rate of NEC, duration of hospitalization and the final in-hospital mortality rates were comparable between these two groups. Conclusions: Probiotic supplementation of extremely preterm infants soon after the initiation of feeding increased the abundance of Lactobacillus. Probiotics may reduce the risk of late-onset sepsis, but further randomized controlled trials are warranted in the future.
Abstract
Background
Streptococcus pneumoniae
is a common cause of post-influenza secondary bacterial infection, which results in excessive morbidity and mortality. Although 13-valent pneumococcal ...conjugate vaccine (PCV13) vaccination programs have decreased the incidence of pneumococcal pneumonia, PCV13 failed to prevent serotype 3 pneumococcal disease as effectively as other vaccine serotypes. We aimed to investigate the mechanisms underlying the co-pathogenesis of influenza virus and serotype 3 pneumococci.
Methods
We carried out a genome-wide screening of a serotype 3
S. pneumoniae
transposon insertion mutant library in a mouse model of coinfection with influenza A virus (IAV) to identify the bacterial factors required for this synergism.
Results
Direct, high-throughput sequencing of transposon insertion sites identified 24 genes required for both coinfection and bacterial infection alone. Targeted deletion of the putative aminotransferase (
PA
) gene decreased bacterial growth, which was restored by supplementation with methionine. The bacterial burden in a coinfection with the
PA
gene deletion mutant and IAV in the lung was lower than that in a coinfection with wild-type pneumococcus and IAV, but was significantly higher than that in an infection with the
PA
gene deletion mutant alone. These data suggest that IAV infection alters host metabolism to benefit pneumococcal fitness and confer higher susceptibility to pneumococcal infection. We further demonstrated that bacterial growth was increased by supplementation with methionine or IAV-infected mouse lung homogenates.
Conclusions
The data indicates that modulation of host metabolism during IAV infection may serve as a potential therapeutic intervention against secondary bacterial infections caused by serotype 3 pneumococci during IAV outbreaks in the future.
Helicobacter pylori
infection is closely associated with various gastrointestinal diseases and poses a serious threat to human health owing to its increasing antimicrobial resistance.
H. pylori
...possesses two major virulence factors, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), which are involved in its pathogenesis. Probiotics have recently been used to eradicate
H. pylori
infection and reduce the adverse effects of antibiotic-based therapies.
Parabacteroides goldsteinii
MTS01 is a novel next-generation probiotic (NGP) with activities that can alleviate specific diseases by altering the gut microbiota. However, the mechanism by which
P. goldsteinii
MTS01 exerts its probiotic effects against
H. pylori
infection remains unclear. Our results showed that administration of
P. goldsteinii
MTS01 to
H. pylori
-infected model mice altered the composition of the gut microbiota and significantly reduced serum cholesterol levels, which mitigated
H. pylori
-induced gastric inflammation. In addition, the pathogenic effects of
H. pylori
VacA and CagA on gastric epithelial cells were markedly abrogated by treatment with
P. goldsteinii
MTS01. These results indicate that
P. goldsteinii
MTS01 can modulate gut microbiota composition and has anti-virulence factor functions, and thus could be developed as a novel functional probiotic for reducing
H. pylori
-induced pathogenesis.
Dissemination of multidrug-resistant, particularly tigecycline-resistant, Acinetobacter baumannii is of critical importance, as tigecycline is considered a last-line antibiotic. Acquisition of ...tet(X), a tigecycline-inactivating enzyme mostly found in strains of animal origin, imparts tigecycline resistance to A. baumannii. Herein, we investigated the presence of tet(X) variants among 228 tigecycline-non-susceptible A. baumannii isolates from patients at a Taiwanese hospital via polymerase chain reaction using a newly designed universal primer pair. Seven strains (3%) carrying tet(X)-like genes were subjected to whole genome sequencing, revealing high DNA identity. Phylogenetic analysis based on the PFGE profile clustered the seven strains in a clade, which were thus considered outbreak strains. These strains, which were found to co-harbor the chromosome-encoded tet(X6) and the plasmid-encoded blaOXA-72 genes, showed a distinct genotype with an uncommon sequence type (Oxford ST793/Pasteur ST723) and a new capsular type (KL129). In conclusion, we identified an outbreak clone co-carrying tet(X6) and blaOXA-72 among a group of clinical A. baumannii isolates in Taiwan. To the best of our knowledge, this is the first description of tet(X6) in humans and the first report of a tet(X)-like gene in Taiwan. These findings identify the risk for the spread of tet(X6)-carrying tigecycline- and carbapenem-resistant A. baumannii in human healthcare settings.
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