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In this study, 23 children were enrolled, aged 4 years and 8 months to 9 years and 5 months, of which one child was 4 years old and 8 months old, and the rest were > 5 years old. The mean age was 5 years and 8 months. No statistical differences in age were observed between AFY group, AF group, and KHJB group (P = 0.361).
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Antibody titers were ≥ 1:160 in the general treatment and probiotics groups. MP nucleic acid levels in the first throat swabs were all positive.
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After high-throughput sequencing of fecal samples from probiotic and healthy control groups, we generated 10,268,989 valid sequences in total. The average number of sequences/sample was 277,540.24.
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Some sequences were randomly selected from our data, the number of species represented by sequences counted, and a dilution curve constructed using sequence and species numbers. As shown (Figure 1), as the sample volume increased, sample flora OTUs tended to be stable and the curve tended to be flat. This indicated that sequencing data were sufficient and the sequencing depth of the sample had been reached.
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OTUs were higher in KHJB group than in AF and AFY groups (P < 0.05). The Chao1 index was higher in the KHJB group than in the AFY group (P < 0.05); no significant differences were observed between KHJB and AF groups. The Shannon index was higher in the KHJB group than in the AF group (P < 0.05); no significant differences were observed between KHJB and AFY groups. The Simpson index was higher in the KHJB group than in the AF group (P < 0.05); no significant differences were observed between KHJB and AFY groups.
The mean number of OTUs in stools was lower in the AF group than in the KHJB group, but differences were not statistically significant. The average Chao1 index of the KHJB group was higher than in the stool sample AF_A, which was higher than in the second stool sample AF_B; however, differences were not statistically significant. The average Shannon index was higher in the KHJB group than in the AF group, but differences were not statistically significant. The average Simpson index was higher in the KHJB group than AF_B which was higher than AF_A; however, differences were not statistically significant.
OTUs in the AFY group were lower than in the KHJB group (P < 0.05). The Chao1 index was higher in the KHJB group than in the AFY group (P < 0.05). The average Chao1 index of the stool sample AFY_B was lower than in AFY_A, but differences were not statistically significant. The average Shannon index was higher in the KHNB group than in the stool sample AFY_B, which was higher than in AFY_A; however, differences were not statistically significant. The average Simpson index of the KHJB group was higher than in the stool sample AFY_B, which was higher than in AFY_A; however, differences were not statistically significant (Figure 2).
Figure 2. Comparison of α-diversity among groups. AF, general treatment group; AF_A, the first stool sample; AF_B, the second stool sample; AFY, probiotic group; AFY_A, the first stool sample; AFY_B, the second stool sample; KHJB, healthy control group. The intragroup differences of Shannon and Simpson index increased after MP infection and reduced after treatment in AFY group.
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PCoA showed that the intragroup differences of bacterial community structures increased in AF and AFY groups comparing to KHJB group, while structural differences between AF and AFY groups were significant (Figure 3). Adonis analysis showed no significant differences in bacterial community structures between AF, AFY, and KHJB groups (R2 = 0.097, P = 0.087).
Figure 3. Principal coordinate analysis (PCoA) plots of individual fecal microbiota based on weighted UniFrac (A) and unweighted UniFrac distances (B). AF, general treatment group; AFY, probiotic group; KHJB, healthy control group.
UPGMA clustering tree analysis based on the Weighted UniFrac distance showed that the bacterial community structures in AFY group are tending to be clustered together after treatment; While similarity in bacterial community structures were observed pre- and post-treatment in AF group. These results suggested that probiotics effectively stabilized intestinal flora (Figure 4).
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In total, ten phyla were detected across all samples: Firmicutes, Proteobacteria, Bacteroides, Actinobacteria, Verrucomicrobia, Patescibacteria, Tenericutes, Euryarchaeota, Fusobacteria, and Epsilonbacteraeota (Figures 5 and 6).
Among groups, the proportion of Verrucomicrobia and Euryarchaeota was lower in the AFY group than in the AF group, and the proportion of Fusobacteria was higher in the AFY group than in the AF group (P < 0.05). When compared with the KHJB group, the proportion of Actinobacteria and Verrucomicrobia decreased, while the proportion of Bacteroidetes and Fusobacteria increased (P < 0.05).
The proportion of Actinomycetes in stool sample AF_B was lower in the AF group than in the KHJB group (P < 0.05). In the AFY group, the proportion of Epsilonbacteraeota was lower in the stool sample AFY_B than in AFY_A (P < 0.05). The proportion of Actinomycetes and Firmicutes in the stool specimen AFY_B was lower than in samples from the KHJB group (P < 0.05).
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The proportion of Enterorhabdus was lower in the AF group than in the KHJB group, while the proportion of Lachnoclostridium was higher in the AF group than in the KHJB group (P < 0.05). The proportion of Akkermansia was lower in the AFY group than in the AF group, and the proportion of Bifidobacteria and Akkermansia was lower in the AFY than in the KHJB group. The proportion of Enterococcus, Lachnoclostridium, Clostridium erysipelas, and Erysipelatoclostridium increased when compared with levels in KHJB group (P < 0.05).
The proportion of Faecalibacterium and Eubacteriumhallii in stool samples AF_A was lower group than in the KHJB group (P < 0.05). The ratio of Bifidobacteria and Romboutsia in stool samples AF_B was lower than in the KHJB group (P < 0.05). In the AFY group, the proportion of Escherichia-Shigella and Butyrivibrio in stool samples AFY_B was lower than in AFY_A. The proportion of Bifidobacteria in stool samples AFY_A was lower than in KHJB group. The proportion of E. coli-Shigella and Subdoligranulum in stool samples AFY_B was lower than in KHJB group. When compared with the KHJB group, the proportion of Enterococcus, Lachnoclostridium, Roseburia, and Erysipelatoclostridium was lower in AFY group (P < 0.05, Figures 7 and 8).
Figure 7. The top 30 different operational taxonomic units (OTUs) in bacterial communities. The graph compares the average proportion of different bacteria in each group and indicates overall changes in bacterial communities. The y-axis represents percentages. On the whole, bacteria were sorted according to the proportion and size of groups. In this figure, they were sorted according to bacteria in the control group.
Figure 8. The top 31–60 different operational taxonomic units (OTUs) in bacterial communities. The graph compares the average proportion of different bacteria in each group and indicates overall changes in bacterial communities. The y-axis represents percentages.
In the probiotic group (AFY), after receiving “combined live Bifidobacterium, Lactobacillus, Enterococcus, and B. cereus tablets”, the proportion of Bifidobacterium, Lactobacillus, and Enterococcus in stool samples AFY_B was higher than that in AFY_A, although differences were not statistically significant (P = 0.454, P = 0.113, and P = 0.463, respectively), but the proportion of Enterococcus in stool specimens (AFY_B) after probiotic treatment increased significantly when compared with healthy controls (P < 0.05). The proportion of Bifidobacterium in pre-treatment stool specimens (AFY_A) was lower than in healthy controls (P < 0.05), but no differences in Bifidobacterium ratios in stool specimens after probiotics treatment were noted when compared with healthy controls (Figure 9).
Figure 9. Bifidobacterium, Lactobacillus, and Enterococcus in probiotic (AFY) and healthy control groups (KHJB).
To analyze the influence of different treatment to intestinal flora, the changes in proportions of bacteria in each individual were analyzed at genus level. Fourteen bacterial species in the general treatment group increased and 21 species decreased after treatment; 13 species in the probiotic treatment group were increased and 21 species decreased (Table 1).
Table 1. The bacteria of inter-individual flora changes after treatment in general treatment (AF) and probiotic treatment groups (AFY)
General treatment group (AF) Probiotics group (AFY) Decreased after treatment Increased after treatment Decreased after treatment Increased after treatment Ruminococcus gnavus Fusicatenibacter Faecalibacterium Blautia Anaerostipes Faecalibacterium Anaerostipes Streptococcus Enterococcus Subdoligranulum Butyricicoccus Hungatella Lachnoclostridium Roseburia Lachnospiraceae Lachnoclostridium Streptococcus Ruminococcaceae Roseburia Peptostreptococcaceae Flavonifractor Veillonella Ruminococcus torques Flavonifractor Hungatella Lachnospira Fusicatenibacter Ruminiclostridium Ruminococcus torques Lachnospiraceae Granulicatella Phascolarctobacterium Ruminiclostridium5 Eubacterium eligens Ochrobactrum Veillonella Sellimonas ChristensenellaceaeR-7 Escherichia-Shigella Micrococcaceae Parabacteroides Ruminiclostridium Parasutterella Actinomyces Parasutterella LachnospiraceaeND3007 Pseudomonas Haemophilus Akkermansia Romboutsia Akkermansia Halomonas Bifidobacterium Pseudomonas Bacteroides Bosea Sellimonas Haemophilus Christensenellaceae Phyllobacterium Ruminiclostridium Ochrobactrum Lachnospiraceae Actinomyces Romboutsia Micrococcaceae Bosea Bacteroides Haemophilus
doi: 10.3967/bes2022.070
Using 16S rDNA Sequencing Technology to Preliminarily Analyze Intestinal Flora in Children with Mycoplasma pneumoniae Pneumonia
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Abstract:
Objective We investigated changes in the intestinal flora of children with Mycoplasma pneumoniae pneumonia (MPP). Methods Between September 2019 and November 2019, stool samples from 14 children with MPP from The Fourth Hospital of Baotou city, Inner Mongolia Autonomous Region, were collected and divided into general treatment (AF) and probiotic (AFY) groups, according to the treatment of “combined Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus cereus tablets live”. High-throughput 16S rDNA sequencing was used to identify intestinal flora. Results Intestinal flora abundance and diversity in children with MPP were decreased. Both Shannon and Simpson indices were lower in the AF group when compared with healthy controls (P < 0.05). When compared with healthy controls, the proportion of Enterorhabdus was lower in the AF group, while the proportion of Lachnoclostridium was higher (P < 0.05). The proportion of Bifidobacteria and Akkermansia was lower in the AFY group but Enterococcus, Lachnoclostridium, Roseburia, and Erysipelatoclostridium proportions were higher. The proportion of Escherichia coli–Shigella in the AFY group after treatment was decreased (P < 0.05). Conclusions The intestinal flora of children with MPP is disturbed, manifested as decreased abundance and diversity, and decreased Bifidobacteria. Our probiotic mixture partly improved intestinal flora disorders. -
Figure 2. Comparison of α-diversity among groups. AF, general treatment group; AF_A, the first stool sample; AF_B, the second stool sample; AFY, probiotic group; AFY_A, the first stool sample; AFY_B, the second stool sample; KHJB, healthy control group. The intragroup differences of Shannon and Simpson index increased after MP infection and reduced after treatment in AFY group.
Figure 7. The top 30 different operational taxonomic units (OTUs) in bacterial communities. The graph compares the average proportion of different bacteria in each group and indicates overall changes in bacterial communities. The y-axis represents percentages. On the whole, bacteria were sorted according to the proportion and size of groups. In this figure, they were sorted according to bacteria in the control group.
Table 1. The bacteria of inter-individual flora changes after treatment in general treatment (AF) and probiotic treatment groups (AFY)
General treatment group (AF) Probiotics group (AFY) Decreased after treatment Increased after treatment Decreased after treatment Increased after treatment Ruminococcus gnavus Fusicatenibacter Faecalibacterium Blautia Anaerostipes Faecalibacterium Anaerostipes Streptococcus Enterococcus Subdoligranulum Butyricicoccus Hungatella Lachnoclostridium Roseburia Lachnospiraceae Lachnoclostridium Streptococcus Ruminococcaceae Roseburia Peptostreptococcaceae Flavonifractor Veillonella Ruminococcus torques Flavonifractor Hungatella Lachnospira Fusicatenibacter Ruminiclostridium Ruminococcus torques Lachnospiraceae Granulicatella Phascolarctobacterium Ruminiclostridium5 Eubacterium eligens Ochrobactrum Veillonella Sellimonas ChristensenellaceaeR-7 Escherichia-Shigella Micrococcaceae Parabacteroides Ruminiclostridium Parasutterella Actinomyces Parasutterella LachnospiraceaeND3007 Pseudomonas Haemophilus Akkermansia Romboutsia Akkermansia Halomonas Bifidobacterium Pseudomonas Bacteroides Bosea Sellimonas Haemophilus Christensenellaceae Phyllobacterium Ruminiclostridium Ochrobactrum Lachnospiraceae Actinomyces Romboutsia Micrococcaceae Bosea Bacteroides Haemophilus -
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