Microbiome comparison in rat groups in relation to chemotherapy and Lactobacillus. This descriptive study investigates the fecal microbiota of rats subjected to chemotherapy (cyclophosphamides for cancer) and significant health gut improvement when co-treated with a cocktail of three Lactobacillus spp. in continuation of our work on Lactobacillus/bacillus on cholesterolemia, lipidemia, diarrhea, and scour [61–62, 66]. Although the sample size is smaller (N = 50), the topic is extremely important in cancer pharmacological treatments because cyclophosphamides (CTX) used against cancerous tumors are frequently found to severely damage the patient’s immune system. We used rats that did not have cancer. They were healthy rats who had been severely harmed by CTX treatments (chemotherapy). The CTX-bioproduct study design includes five different experimental groups: healthy control rats (1), treated rats (CTX chemotherapy) that were either only treated with CTX (2) or treated with CTX and a low (3), middle (4) or high (5) complement dose of Lactobacillus spp. (Fig. 1). In a previous study, leukocyte concentration, CD4/CD8, interleukin, and TNF-alpha expression were measured in each group (1–5), showing that CTX has a significant effect on the rat immune system [see 65]. Here, the microbiome was assessed in each group (1–5) using ITS and 16S rRNA gene sequencing on the Illumina MiSeq platform, suggesting that health can be maintained despite CTX when using Lactobacillus (Figs. 2–5 & S1-S13 and Tables 1–2 & S1-S4).
Table 2
Mycobiome (Phylum, Class, Order, Family, and Genus of fungi and yeasts) composition in relation to Lactobacillus and cyclophosphamide treatment in five groups of rats. CK: control healthy conditions; IM: immune-attacked (CTX); L: CTX + 3L-low dose; M: CTX + 3L-middle dose; H: CTX + 3L-high dose. Treatment for Lactobacillus: tritherapy (3L) = L. acidophilus SD65 + L. casei SD07 + L. plantarum SD02. The group’s dominant genera are highlighted in bold. * displays a significant increase in specific microbes in CK and the three Lactobacillus group doses (L, M and H). ° shows a marked reduction in specific microbes caused by CTX chemotherapy.† shows increased microbes under chemo (see IM), but not under chemotherapy + Lactobacillus conditions (see L, M and H).
CK
|
IM
|
H
|
M
|
L
|
Phylum
|
|
|
|
|
Ascomycota
|
Ascomycota°
|
Ascomycota
|
Ascomycota
|
Ascomycota
|
Basidiomycota*
|
Basidiomycota°
|
Basidiomycota*
|
Basidiomycota*
|
Basidiomycota*
|
Blastocladiomycota
|
|
|
|
|
Chytridiomycota
|
|
|
|
|
Glomeromycota
|
|
|
|
|
Kickxellomycota
|
Kickxellomycota°
|
Kickxellomycota
|
Kickxellomycota
|
Kickxellomycota
|
Mortierellomycota*
|
Mortierellomycota°
|
Mortierellomycota*
|
Mortierellomycota*
|
Mortierellomycota*
|
Mucoromycota
|
Mucoromycota†
|
Mucoromycota
|
Mucoromycota
|
Mucoromycota
|
Olpidiomycota
|
Olpidiomycota†
|
Olpidiomycota
|
Olpidiomycota
|
Olpidiomycota
|
Class
|
|
|
|
|
Agaricomycetes
|
|
|
|
|
Agaricostilbomycetes*
|
Agaricostilbomycetes°
|
Agaricostilbomycetes*
|
Agaricostilbomycetes*
|
Agaricostilbomycetes*
|
Blastocladiomycetes
|
|
|
|
|
Cystobasidiomycetes
|
|
|
|
|
Dothideomycetes
|
Dothideomycetes°
|
Dothideomycetes
|
Dothideomycetes
|
Dothideomycetes
|
Eurotiomycetes
|
Eurotiomycetes°
|
Eurotiomycetes
|
Eurotiomycetes
|
Eurotiomycetes
|
Exobasidiomycetes
|
|
|
|
|
Leotiomycetes*
|
Leotiomycetes°
|
Leotiomycetes*
|
Leotiomycetes*
|
Leotiomycetes*
|
Malasseziomycetes
|
|
|
|
|
Microbotryomycetes
|
|
|
|
|
Mortierellomycetes
|
|
|
|
|
Mucoromycetes
|
Mucoromycetes†
|
Mucoromycetes
|
Mucoromycetes
|
Mucoromycetes
|
Olpidiomycetes
|
|
|
|
|
Pezizomycetes
|
|
|
|
|
Rhizophlyctidomycetes
|
|
|
|
|
Saccharomycetes
|
Saccharomycetes°
|
Saccharomycetes
|
Saccharomycetes
|
Saccharomycetes
|
Sordariomycetes
|
Sordariomycetes°
|
Sordariomycetes
|
Sordariomycetes
|
Sordariomycetes
|
Tremellomycetes*
|
Tremellomycetes°
|
Tremellomycetes*
|
Tremellomycetes*
|
Tremellomycetes*
|
Ustilaginomycetes
|
|
|
|
|
Wallemiomycetes
|
|
|
|
|
Order
|
|
|
|
|
Agaricales
|
|
|
|
|
Capnodiales*
|
Capnodiales°
|
Capnodiales*
|
Capnodiales*
|
Capnodiales*
|
Cystofilobasidiales*
|
Cystofilobasidiales°
|
Cystofilobasidiales*
|
Cystofilobasidiales*
|
Cystofilobasidiales*
|
Eurotiales
|
Eurotiales°
|
Eurotiales
|
Eurotiales
|
Eurotiales
|
Filobasidiales
|
|
|
|
|
Glomerellales*
|
Glomerellales°
|
Glomerellales*
|
Glomerellales*
|
Glomerellales*
|
Helotiales
|
|
|
|
|
Hypocreales
|
Hypocreales°
|
Hypocreales
|
Hypocreales
|
Hypocreales
|
Malasseziales
|
Malasseziales
|
Malasseziales*
|
Malasseziales*
|
Malasseziales*
|
Microascales
|
|
|
|
|
Mortierellales
|
|
|
|
|
Mucorales
|
Mucorales†
|
Mucorales
|
Mucorales
|
Mucorales
|
Olpidiales
|
|
|
|
|
Pleosporales
|
|
|
|
|
Saccharomycetales
|
Saccharomycetales°
|
Saccharomycetales
|
Saccharomycetales
|
Saccharomycetales
|
Sordariales
|
|
|
|
|
Tremellales
|
|
|
|
|
Trichosporonales
|
Trichosporonales°
|
Trichosporonales
|
Trichosporonales
|
Trichosporonales
|
Ustilaginales
|
|
|
|
|
Wallemiales
|
|
|
|
|
Family
|
|
|
|
|
Aspergillaceae
|
Aspergillaceae°
|
Aspergillaceae
|
Aspergillaceae
|
Aspergillaceae
|
Cordycipitaceae*
|
Cordycipitaceae°
|
Cordycipitaceae*
|
Cordycipitaceae*
|
Cordycipitaceae*
|
Debaryomycetaceae
|
|
|
|
|
Didymellaceae
|
Didymellaceae°
|
Didymellaceae
|
Didymellaceae
|
Didymellaceae
|
Hypocreaceae
|
|
|
|
|
Lichtheimiaceae
|
|
|
|
|
Malasseziaceae
|
|
Malasseziaceae*
|
Malasseziaceae*
|
Malasseziaceae*
|
Metschnikowiaceae
|
|
|
|
|
Microascaceae
|
|
|
|
|
Mucoraceae
|
Mucoraceae†
|
Mucoraceae
|
Mucoraceae
|
Mucoraceae
|
Mycosphaerellaceae*
|
Mycosphaerellaceae°
|
Mycosphaerellaceae*
|
Mycosphaerellaceae*
|
Mycosphaerellaceae*
|
Nectriaceae
|
Nectriaceae°
|
Nectriaceae
|
Nectriaceae
|
Nectriaceae
|
Plectosphaerellaceae
|
|
|
|
|
Pleosporaceae
|
Pleosporaceae°
|
Pleosporaceae
|
Pleosporaceae
|
Pleosporaceae
|
Pichiaceae
|
Pichiaceae°
|
Pichiaceae
|
Pichiaceae
|
Pichiaceae
|
Rhizopodaceae*
|
Rhizopodaceae°
|
Rhizopodaceae*
|
Rhizopodaceae*
|
Rhizopodaceae*
|
Thermoascaceae*
|
Thermoascaceae°
|
Thermoascaceae*
|
Thermoascaceae*
|
Thermoascaceae*
|
Trichosporononaceae*
|
Trichosporononaceae°
|
Trichosporononaceae*
|
Trichosporononaceae*
|
Trichosporononaceae*
|
Ustilaginaceae
|
|
|
|
|
Genus
|
|
|
|
|
Alternaria
|
Alternaria°
|
Alternaria
|
Alternaria
|
Alternaria
|
Aspergillus
|
Aspergillus°
|
Aspergillus
|
Aspergillus
|
Aspergillus
|
Candida
|
|
|
|
|
Chlamydomyces*
|
Chlamydomyces°
|
Chlamydomyces*
|
Chlamydomyces*
|
Chlamydomyces*
|
Fusarium
|
Fusarium°
|
Fusarium
|
Fusarium
|
Fusarium
|
Kodamaea
|
Kodamaea°
|
Kodamaea
|
Kodamaea
|
Kodamaea
|
Lecanicillium*
|
Lecanicillium°
|
Lecanicillium*
|
Lecanicillium*
|
Lecanicillium*
|
Malassezia
|
|
|
|
|
Meyerozima
|
|
|
|
|
Mucor
|
Mucor†
|
Mucor
|
Mucor
|
Mucor
|
Mycosphaerella
|
Mycosphaerella°
|
Mycosphaerella
|
Mycosphaerella
|
Mycosphaerella
|
Pichia
|
Pichia°
|
Pichia
|
Pichia
|
Pichia
|
Penicillium
|
|
|
|
|
Rhizomucor*
|
Rhizomucor°
|
Rhizomucor*
|
Rhizomucor*
|
Rhizomucor*
|
Rhizopus*
|
Rhizopus°
|
Rhizopus*
|
Rhizopus*
|
Rhizopus*
|
Sarocladium*
|
Sarocladium°
|
Sarocladium*
|
Sarocladium*
|
Sarocladium*
|
Simplicillium
|
|
|
|
|
Talaromyces
|
|
|
|
|
Trichoderma
|
|
|
|
|
Xerochrysium
|
Xerochrysium†
|
Xerochrysium
|
Xerochrysium
|
Xerochrysium
|
The Venn Diagram depicting the relationships between the five groups in relation to chemotherapy and Lactobacillus suggested a relationship between healthy control and CTX + increasing dose of Lactobacillus spp. (CK and M-H; Figure S1). The position, configuration, and overlap of the circles indicating the relationships between the groups showed a gradual increase in gut flora and overall health depending on the Lactobacillus spp. complement dose (L-to-H; Figure S1). The IM group (CTX alone) was further along the bottom representing a higher number of total unique OTUs (17.24%), similarly to L dose (number of total unique OTUs: 15.82%). The Venn Diagram showed that the core microbiomes in the H and control groups were related when we looked at the number of total unique OTUs (10.52–10.82%), whereas M complement doses of Lactobacillus spp. remained too closely related to CTX alone conditions (15.07%; Fig. 2A). The middle complement dose (×2, two-fold) fell between the control (CK) and immune-damaged (IM) groups (Figure S1). The H-dose group was opposite the IM group, with no overlap with the control group (CK). As a result, increasing the Lactobacillus concentration (more than five-fold) appeared to be required to produce an even more significant beneficial effect on the microbiome of rats treated by CTX (Figure S1).
The five groups appear to have relatively similar levels of similarity on Venn diagrams (Figure S1). All samples (three per group) and OTU numbers were consistent across the groups CK-H. In each group, approximately the same number of ITS and 16S sequences were obtained in different biological replicates (Table S1). The five different groups obtained roughly the same sequence quantity (Table S1). There were no differences in the number of OTUs found at each taxonomic level (phylum, class, order, family, genus, and species; Tables S2 & S3). Although all of the OTUs could be classified, the classification of OTUs at different taxonomic levels in five rat groups related to chemotherapy and Lactobacillus treatment revealed no discernible differences in OTU counting (Tables S2 & S3). This was seen in both grouped (CK, IM, L, M and H) and ungrouped (C101, C103, C105, IM015, IM021, IM024, L102, L103, L104, M201, M202, M203, H105, H204 and H205) samples (Tables S2 & S3). As a result, the sample sizes were similar, particularly for Family and Genus (Figure S2). Using the OTU table for sample diversity in PCA, rank abundance curve, NMDS, principle coordinate analysis (PcoA), Bray-Curtis distance plot (default semimetric), binary Jaccard distance matrix (metric), and UPGMA, however, H was clustered with CK.
The PCA based on OTU composition revealed significant differences in the five groups (Fig. 2AB). At the community level, PCA revealed similarities between the chemotherapy + high lactobacillus spp. and control groups, with Fusarium, Talaromyces, Sarocladium, Aspergillus, and Mucor falling outside a common spectrum of microbe genera (Fig. 2A).
Orthogonal Projections to Latent Structures and Discriminant Analysis (OPLS-DA) showed Mucor and Talaromyces to be associated with ill conditions (chemotherapy alone), but a wide range of microbes to be associated with control and chemotherapy + high Lactobacillus conditions (Fig. 2B). The OPLS-DA analysis showed that the distances between the CK and H groups were very small, while the L, M, and IM groups were clearly separated. Fusarium, Sarocladium, Kodamaea, Verticillium, Candida, and Chlamydomyces were among the fungi with overlapping distributions in the two groups, CK and H. Trichoderma, Acremonium, and Malassezia were more closely associated with the Lactobacillus groups H, M, and L. Pichia and Aspergillus were separated from this group but mixed with the CK control group. Microbial fungi such as Mucor and Talaromyces, as well as Xeromyces, Xerochrysium and Penicillium, were associated with CTX and the immune-damage group (Fig. 2AB).
In addition to PCA, CK-samples tended to cluster with H-, M- and L-samples in NMDS (Fig. 3). The IM group differed significantly from the other groups, with clear mean differences between microbiomes from CTX-treated samples and those from controls (no treatment) and rats treated with Lactobacillus in addition to CTX (Bray-Curtis; Fig. 3A). The NMDS graph using the Jaccard index collapsed very clear information: CK grouped with H-samples, showing mean similarities between H-microbiomes and controls (Jaccard; Fig. 3B). Using PcoA as a principle analysis, the same grouping was observed, lending support to PCA and NMDS (Fig. 4). Both the Bray-Curtis (abundance) and Jaccard (0/1 data) indices showed a pair of communities with comparable species richness (H and CK; Fig. 4). The similarity between the CK and H samples was confirmed by unweighted and non-metric MDS analysis (UPGMA). H branches clustered with CK with a low distance value (0.005 − 0.0028) on the UPGMA tree (Figure S3A). There were also unweighted pairs found between M and L groups (distance value 0.031–0.111, Figure S3A; 0.003, Figure S2B). The branches representing the immune-damaged group (IM) clustered at the bottom of the tree, indicating the mean distance (or difference) of IM compared to CK, H, M, and L (Figure S3). Therefore, many of our results (PCA, PcoA, NMDS, and UPGMA) do show Lactobacillus-control clustering, but even in these plots, it is difficult to see how much closer the high Lactobacillus group is to controls when compared to IM because of the large outlier within the IM grouping (Figs. 2–5 & S3). We couldn’t conclude that the control and Lactobacillus groups had more microbial diversity, but in our rank abundance curve (i.e. species richness and species evenness), the IM group (CTX alone) was further along the x axis, representing a higher number of total unique OTUs (Figure S4). We used Specaccum (number of species vs number of samples) to show how species richness increased as sample size increased. The curve rapidly reached a plateau. There were no significant differences in species richness when increasing the number of samples (up to 12) lumped into a single analysis, which was not done on a per group basis (Figure S5). In grouped and ungrouped samples, the Chao1, Simpson, Shannon, Pielou_e, observed species and Goods_coverage indices (alpha-diversity) were calculated (Figure S6 & Table S4). These indices (Chao1, Pielou_e and observed_species) indicated that the CK and H groups had similar community richness and species evenness (Figure S6 & Table S4). The goods_coverage index showed significant differences between H and IM. Goods_coverage metrics at OTU levels (sample completeness, p = 0.76) showed a high level of microbial diversity in H (Figure S6 & Table S4).
Mycobiome of five different groups of rats profiled in relation to chemotherapy andLactobacillus. Examining of individual taxon abundance using heatmaps was useful to analyze fungal taxa clustering based on the abundance of each fungus in the five rat groups (Figs. 5 & S7). A heatmap analysis of fungi revealed the relative abundance of each taxon in CK, IM, H, M, and L. In this descriptive study, Table 1 lists the various fungal taxa found in the CK, CTX, and 3L groups. From the Acaulium (syn. Scopulariopsis) genus to Xeromyces, several broad types of microbes were identified in the CK group (Fig. 5 & Table 1). CK (control, healthy condition) had high levels of Candida, Cutaneotrichosporon, Filobasidum, Fusarium, Kernia, Kodamaea, Lecanicillium, Meyerozima, Papiliotrema (Cryptococcus), Pichia, Rhodoturula, Verticillium, and Wallemia, whereas IM (immune-attacked) had high levels of Acaulium, Mucor, Olpidium, Penicillium, Periconia, Phallus, Xerochrysium, and Xeromyces (Fig. 5 & Table 1). However, treating rats with Lactobacillus in addition to Cyclophosphamide increased relative fecal abundance of many different fungal taxa, including Acremonium, Aspergillus, Chlamydomyces, Fusarium, Mortierella, Phallus, Rasamsonia, Rhizophlyctis, Rhizopus, Talaromyces, and Trichoderma (in H group), Coprinellus, Microascus, Mycosphaerella, Phialocephala, Pseudogymnoascus, Rhizomucor, Rhizophlyctis, Rhizopus, Sarocladium, Scytalidium, Thermoascus, and Ustilago (in M group), and Mallassezia, Plectosphaerella, Rhizophlyctis, Rhizopus, Saccharomyces, Simplicillium, Sodiomyces, and Tausonia (in L group; Fig. 5 & Table 1). As a result, among the regulated fungi are species that are not known as animal commensals or pathogens. Lecanicillium fungi are classified as generalist entomopathogenic fungi [98]. Ustilago is a Poaceae plant pathogen [99]. Phallus mushrooms are big saprotrophic mushrooms [100]. However, the presence of these fungi in the rat microbiota is not necessarily suspect and may merit further investigation. The breeding history of rats takes place in the Institute of Medicine’s Class II animal facility in SAMS (Specific Free Pathogen/SPF facilities and acute hospital care settings that are designed to keep organisms in sterile environments). Saprophytic basidiomycetes are well-known wood-decaying fungi, but Phallus sequences have been found in animal penis and urethra, where they play a role in male fertility [101]. In fact, little is known about the fungal flora of rodent’s digestive and reproductory tracts. Lecanicillium species are pathogens that parasitize not only insects but also worms and many other fungi, which could explain their presence in gut fungi associated with rats. Lecanicillium strains have been found in gut fungi associated with marmots [102]. A large variety of ‘forgotten’ odd fungi, including Ustilaginales and Ustilago sp., are found in the human digestive tract [103–106], as seen in rodents (Fig. 5 & Table 1). So it is not surprising that fungal sequences like Lecanicillium, Phallus, and Ustilago have been found in the fecal DNA of CTX-rat models. It has been described in a variety of other animal species, including humans. What is more unusual or surprising is the discovery that these fungi are differentially regulated by CTX and/or 3L conditions, which is a critical key point in addressing their prevalence in the gut microbial system (Fig. 5 & Table 1).
Phallus was found in cyclophophasmide-treated rats and rats treated with CTX + high 3L lactobacilli in the heatmap (Fig. 5). Despite CTX treatment, medium and low doses of 3L were able to eradicate Phallus fungi, as shown by triplicate samples (Figure S7 & Table 1), even though Phallus infection was not prevalent in all IM samples (Figure S7). More interestingly, the heatmap analysis highlighted two taxa in particular, Fusarium and Pichia, which were found in high abundance in feces from control and high lactobacillus-treated groups and samples or were repeatedly found in control samples but significantly altered by chemotherapy (Figs. 5 & S7). Furthermore, there was a correlation between Rhizopus and Lactobacillus treatment. Chemotherapy signifcantly reduced Rhizopus-levels, but increased when Lactobacillus was added to phosphamide. Rhizopus was found in abundance in H, M, and L groups (Figs. 5 & S7). The Rhizopus microbe was more abundant in M samples (CTX + middle dose/2.5 ml/kg bodyweight of Lactobacillus), indicating that a specific dose of bioproduct should be chosen for effective regulation of specific microbes (Figs. 5, S7 & Table 1). We found that high-, medium-, and low-dose 3L cocktail gavages were effective in lowering Acaulium, Mucor, Olpidium, Penicillium, Periconia, Xerochrysium, and Xeromyces levels (Figs. 5, S7 & Table 1).
The microbial composition distribution histograms of each sample were displayed at the phylum, order, class, family, and genus levels (Fig. 6 & Table 2) in our descriptive analysis of rats in relation to CTX and Lactobacillus (N = 50; analysis of groups and individual samples, the same chemotherapy session, one drug, five shots, addition of Lactobacilli, 3L-test, three different doses, comparison with controls, beneficial effects analysis). The dominant microbial phyla were similar in control and 3L therapy conditions (Fig. 6A & Table 2). CTX, on the other hand, caused a significant decrease in Ascomycota-levels, which was not seen with high-doses of 3L during chemotherapy (Fig. 6A). Many other microbial fungal phyla, including Basidiomycota, Kickxellomycota, and Mortierellomycota benefited from 3L gavage (Fig. 6A & Table 2). Mucoromycota and Olpidiomycota levels in rat fecal microbiomes increased during CTX chemotherapy but remained low when H, M, or L doses of Lactobacillus were added to CTX (Fig. 6A & Table 2). Similarly, analysis of the distribution of microbial fungal classes, orders, families, and genera in the five rat groups showed specific beneficial effects of Lactobacillus treatment in addition to cancer chemotherapy (Fig. 6B-E & Table 2). Dothideomycetes, Eurotiomycetes, Saccharomycetes, Sordariomycetes, and Tremellomycetes were the most abundant microbial fungal classes in healthy control rats without any other treatment than normal saline injection. CTX chemotherapy had a significant impact on all five classes. Chemotherapy also increased Mucoromycetes levels in the fecal microbiome. With high dose injections of 3L, Dothideomycetes, Eurotiomycetes, and Sordariomycetes were kept at normal levels. Mucoromycetes were kept at normal levels in all three Lactobacilli-treated samples. Agaricostilbocytes, Leotiomycetes, and Tremellomycetes, were also recovered at normal levels after probiotic treatments (Fig. 6B & Table 2). Eurotiales, Hypocreales, Saccharomycetales, and Trichosporonales were the most abundant microbes on an order level not only in CK, but also in H group. The IM group had a different microbial order profiling, with significantly altered levels of Eurotiales, Hypocreales, Saccharomycetales, and Trichosporonales, as well as significantly increased levels of Mucorales. Lactobacillus treatment restored normal levels of Capnodiales, Cystofilobasidiales and Glomerellales that had been affected by chemotherapy. Lactobacillus H, M, and L doses were effective in controlling Mucorales levels. A low dose of Lactobacillus was also particularly effective in stimulating Mallasseziales levels, emphasing the importance of controlling Lactobacillus dose to target specific microbial orders (Fig. 6C & Table 2). Four major microbial families were identified in rat fecal samples related to CTX and 3L therapy: Aspergillaceae, Didymellaceae, Nectriaceae, and Pleosporaceae. Surprizingly, these microbial families were vulnerable to chemotherapy alone, but were kept alive by combining 3L with CTX-chemo treatment (Fig. 6D). To maintain the levels of Aspergillaceae, a gradual increase of 3L seemed to be required (Fig. 6D). CTX also reduced the levels of Cordycipitaceae, Mycosphaerellaceae, Rhizopodaceae, Thermoascaceae, and Trichosporonaceae, but these levels were maintained when CTX was combined with Lactobacillus gavage. This was not true for all of the microbial families found in rat feces. Pichiaceae was one of the microbial families that were down-regulated after CTX treatment, which could not be reversed by adding Lactobacillus during chemo-treatment (Fig. 6D). However, Lactobacillus at high, medium, and low doses had a clear beneficial effect on Mucoraceae control (Fig. 6D). Mucoraceae-levels in the IM group were extremely high, which could be reversed by adding H, M, or L doses of Lactobacillus (Fig. 6D). A low dose of the bioproduct was found to induce especially high levels of Mallasseziaceae (Fig. 6D & Table 2). Alternaria, Aspergillus, Fusarium, and Mycosphaerella were the main microbial genera characteristic of the CK and H groups, respectively, while Mucor was a diagnosis of immune-damage caused by CTX treatment. The addition of Lactobacillus to chemotherapy effectively controlled Mucor. Mucor-levels were found to be extremely low in the H, M, and L groups of rats related to Chemo + Lacto treatment. On Xerochrysium, similar effects were observed. Xerochrysium-levels rose during chemotherapy, but were kept under control by using Lactobacillus at low, medium, and high doses. Other microbial genera such as Chlamydomyces, Lecanicillium, Rhizomucor, and Sarocladium were maintained by Lactobacillus at low, medium, or high doses. Only Pichia was not maintained by Lactobacillus treatment, regardless of the dose of 3L bioproduct (Fig. 6E & Table 2).
When all triplicates were compared (Figure S8), Ascomycota levels were found to be remarkably high in CK and H triplicates (Figure S8A). In contrast, Ascomycota levels were particularly low in IM021, L103, and M201. Mucoromycota and Olpidiomycota were abundant in IM015 samples (Figure S8A). Mucoromycota and Olpidiomycota were significantly lower in Lactobacillus samples, particularly H (H105, H204, and H205). Mortierellomycota were missing in IM triplicates (IM015, IM021, and IM024), but present in C101, C103, L104, M202, M203, H105, H204, and H205 (Figure S8A). High levels of Sordariomycetes, Saccharomycetes, and Tremellomycetes were found in control triplicates (C101, C103, and C105) in ungrouped samples. Sordariomycetes and Tremellomycetes levels remained high in L102, L104, M202, M203, H105, H204, and H205. Eurotiomycetes were found in very low concentrations in IM021. Mucoromycetes were found in abundance in IM015 sample. Eurotiomycetes and Mucoromycetes were kept to normal conditions in all H samples (Figure S8B). On the order level, IM015 was distinguished by a high Mucorales/low Saccharomycetales ratio (Figure S8C). Despite the fact that Saccharomycetales remained low in all H, M, and L Lactobacillus-treated samples, Mucorales levels in Lactobacillus samples were comparable to controls (Figure S8C). Furthermore, Capnodiales levels in medium and high Lactobacillus samples M202-H205 were comparable to those found in C101, C103, and C105. Glomerellales levels were high in both the control (C101 and C103) and Lactobacillus (L104) samples (Figure S8C). Microbial family profiling was diverse in all samples, but particularly in the CK and H groups. C101, C103, C105, H105, H204, and H205 all showed high levels of Nectriaceae and Trichosporonaceae, as well as a variety of other families ranging from Aspergilaceae to Microascaceae. Notably, none of the Lactobacillus samples had the high levels of Mucoraceae found in IM015 (Figure S8D). In the genus taxa summary from ungrouped samples, IM015 had high levels of Mucor, whereas C101, C102, C105, L102, L104, M202, M203, H105, H204, and H205 had high levels of Fusarium but no Mucor to the extent seen in IM105 (Figure S8E). As a result, ungrouped samples of CTX-related rat fecal microbiomes and the effects of adding specific bioproducts also argued for Lactobacillus rather beneficial role in maintaining host health microbiome during chemotherapy.
The analysis of metagenome sequence data (CK versus M) revealed a pattern that overlapped with enriched core microbes in the order Trichosporonales and the phylum Basidiomycota (Figure S9). The relative abundance of Fungi, Ascomycota, Sordariomycetes, Hypocreales, Nectriaceae, and Fusarium in CK and H class samples was very high (above 60000–140000). In the IM, L, and M classes of samples, the relative abundance of Fusarium fungi was less than 50000 (Figure S10A). Fusarium was identified as a key biomarker (i.e, a key community member) of the CK group by LEfSe (LDA, Krustal-Wallis and Wilcoxon; Figure S10B). Comparative metagenomics and network analysis at the phylum level showed a high degree of similarity between control and Lactobacillus-treated rat fecal samples, as well as the dominance of Ascomycota in this network (Figure S11). This CK-Lactobacillus group is not associated with IM samples (in blue; Figure S11A). Mucoromycota (in orange) dominated in CTX- immune-attacked ill rat feces (Figure S11B).
CTX and CTX + Lactobacillus therapy effects on bacteriome and metabolic pathways. The relative abundance of each functional category (biosynthesis, degradation/utilization/assimilation, generation of precursor metabolite and energy, glycan pathways and metabolic clusters) was calculated using pathway abundance and read count abundance (MetaCyc; Figure S12). Differential abundance was found primarily for respiration, fermentation, fatty acid/lipid/carbohydrate degradation, and biosynthetic pathways (Figure S12A). Similarly, in MetaCyc, raw counts for metabolic pathways and enzymes, metabolites, and reaction orthologs revealed a strong statistical significance of differential abundance, primarily for cofactor, prosthetic group, electron carrier, vitamin, fatty acid, and lipid biosynthesis (Figure S12B). Some metabolic pathways in the MetaCyc database can be labeled with a low-level bacterial taxon [107]. As a result, we used MetaCyc to find metabolic pathways and/or bacterial taxa that are specifically related to the five groups of rats for chemotherapy (Figs. 7 & S13). A specific pathway (PWY-7839), 6-hydroxymethyl-dihydropterin diphosphate biosynthesis I, which converts GTP into pterin precursors (methanopterin and sarcinapterin) for the biosynthesis of several cofactors in specific bacterial strains, was found to be particularly highly expressed in CK and Lactobacillus-treated samples due to an increase in S24-7 Muribaculaceae, Prevotella, Clostridiales, Bacteroides, and CF231 Paraprevotellaceae (Fig. 7A). Treatments with M- and H-doses were clearly effective in increasing the levels of Bacteroides and Prevotella, both of which are essential in the pyridoxine pathway required for vitamin B6 synthesis (PYRIDOXSYN-PWY, pyridoxal 5’-phosphate (PLP) biosynthesis I; Fig. 7B). Furthermore, Lactobacillus treatment restored the abundance of helicobacterial taxa required for the TCA cycle (tricarboxylic acid cycle) or the Krebs cycle. Despite chemotherapy, with Lactobacillus treatment, not only Helicobacter-levels, but also “Flexispira” (in purple), Rothia, and Halomonas levels, were maintained (Fig. 7C). Lactobacillus L-, M-, or H-doses, had no effects on Bacillales. To control Halomonas, a high dose of 3L (5.0 ml/kg) was strictly required (Fig. 7C). In IM samples, a formaldehyde oxydation peak was observed. This was linked to the emergence of Enterococcus bacteria in immune compromised conditions (Fig. 7D). Many Enterococcus species are known to be commensals and are not actively causing infection. In our case (Fig. 7D), our results show a peak of Enterococcus linked to chemotherapy (CTX alone), implying that Enterococcus is an active infection. The addition of Lactobacillus to chemotherapy completely eliminated it; no Enterococcus peak was observed in CK, H and M groups (Fig. 7D). TCA-GLYOX-BYPASS, the superpathway or bypass that integrates the common prokaryotic Krebs cycle (TCA) with the glyoxylate shunt, benefited from Lactobacillus treatment during chemotherapy (+ CTX). In both CK and H-dose conditions, a high diversity of bacterial taxa was observed. Enrichment of Enterobacteriaceae, Rothia, Cupriavidus, Halomonas, and Devosia was detected in controls and persisted during CTX chemotherapy when an additive probiotic treatment with high doses of 3L was used. Treatment with Lactobacillus was ineffective on Bacillales at 1.25-5 ml/kg doses (Fig. 7E). Lactobacillus at a medium-dose (2.5 ml/kg) was particularly effective in stimulating Enterobacteriaceae (Fig. 7E). Lactobacillus at a high-dose (5 ml/kg) was particularly effective in stimulating Devosia (Fig. 7E). Similarly, the additive Lactobacillus treatment positively regulated Bacteroidales, Bacteroides, Enterobacteriaceae, Halomonas, and Devosia responsible for (prokaryotic) TCA cycle I (Fig. 7F). CTX chemotherapy and/or treatment with 3L bioproduct had a significant impact on tRNA charging and microbiome. We observed the main stimulatory effects of 3L on S24-7, Prevotella, Bacteroides, Ruminococcus, CF231, and Oscillospira using high doses of Lactobacillus (Fig. 7G). Finally, Illumina and MetaCyc analyses revealed that a middle dose of Lactobacillus had a strong effect on Enterobacteriaceae, which mediate the bacterial superpathway of coenzyme Q ubiquinol-8 biosynthesis (UBISYN-PWY, Fig. 7H).
When other types of metabolic pathways were examined (MetaCyc), the effects of Lactobacillus in addition to CTX were less obvious (Figure S13). No particular bacteria were found for the MetaCyc L-methionine salvage cycle III (PWY-7527, Figure S13A). Lactobacillus doses (M and H) primarily stimulated the anaerobic pathway for oleate biosynthesis IV (PWY-7664), however, the IM group had one sample that was much higher in the abundance of this pathway than all the H Lactobacillus group. The medium group appeared to have higher overall levels than the high group, possibly indicating an effect of 3L on this pathway (Prevotella and Bacteroides) but making any dose response relationship difficult to determine (Figure S13B). M- and H-doses of the bioproduct apparently had similar beneficial effects on mycolate biosynthesis (PWYG-321), with high levels of Bacteroides accumulating in M-treated samples (Figure S13C). Analysis of bacterial strains involved in the pathway teichoic acid (poly-glycerol) biosynthesis, which is part of cell wall biogenesis, seemed to have a positive effect of 3L bioproduct (M and/or H) as an additive to chemotherapy. Lactobacillus contributed to the low levels of Clostridiales, Mogibacteriaceae, Ruminococcaceae, and Gemella, while strains such as Jeotgalicoccus were stimulated (Figure S13D). Except for enterobacter in some low-dose Lactobacillus samples, no specific bacterial strains were identified for the superpathway of L-threonine metabolism (Figure S13E). Chemotherapy (immune-attacked; IM) reduced the levels of Clostridiales, Ruminococcaceae, Ruminococcus, and Oscillospira in the pathway UDP-N-acetyl-D-glucosamine biosynthesis I (UDPNAGSYN-PWY), which could be avoided by combining CTX with a high dose (5 ml/kg) of Lactobacillus (Figure S13F).