Meloidogyne incognita are one of the most destructive pathogens, causing severe losses to tobacco productivity and quality. However, the underlying resistance mechanism of tobacco to M. incognita is not clear. In this study, two tobacco genotypes, K326 and Changbohuang, which are resistant and susceptible to M. incognita, respectively, were used for RNA-sequencing analysis. An average of 35 million clean reads were obtained. Compared with their expression levels in non-infected plants of the same genotype, 4354 and 545 differentially expressed genes (DEGs) were detected in the resistant and susceptible genotype, respectively, after M. incognita invasion. Overall, 291 DEGs, involved in diverse biological processes, were common between the two genotypes. Genes encoding toxic compound synthesis, cell wall modification, reactive oxygen species and the oxidative burst, salicylic acid signal transduction, and production of some other metabolites were putatively associated with tobacco resistance to M. incognita. In particular, the complex resistance response needed to overcome M. incognita invasion may be regulated by several transcription factors, such as the ethylene response factor, MYB, basic helix–loop–helix transcription factor, and indole acetic acid–leucine-resistant transcription factor. These results may aid in the identification of potential genes of resistance to M. incognita for tobacco cultivar improvement.Root-knot nematodes  Meloidogyne incognita are one of the most destructive pathogens, limiting plant productivity and quality. At present, M. incognita is controlled in crops by using chemical nematicides, adequate cultural practices, and soil fumigants. However, using cultivars with resistance or tolerance genes has proved to be the most effective and environmentally friendly strategy for managing M. incognita [Mae and Mag genes in peanut [Mi genes in tomato [Me3 genes in pepper [Nicotianatabacum L.) is not only a model plant for molecular research but also an important economic crop in approximately 120 countries worldwide [M. incognita infection. The resistance to M. incognita varies considerably among tobacco varieties, and exploring the underlying molecular mechanisms of resistance to M. incognita may facilitate the breeding efforts for enhanced-resistance genotypes. Plants have evolved various basal and inducible mechanisms to withstand the invasion by nematodes. Generally, the mechanisms underlying the resistance response in plants involve cell wall modification, proteins with antimicrobial activities, and secondary metabolites, e.g., phytoanticipins and phytoalexins [Root-knot nematodes (RKNs) ncognita . Some prn peanut , Mi genen tomato , and Me3n pepper . Howevern pepper . Tobaccoorldwide . Howeveroalexins . The plaoalexins . Once neoalexins . Resistance to RKNs, mediated by hypersensitivity reaction, is characterized by the production of reactive oxygen species (ROS), overaccumulation of which—also known as oxidative burst—usually causes cells to constantly live in an oxidizing environment, eventually leading to cell death . To overM. incognita infestation in Phaseolusvulgaris [Cucumismetuliferus [Solanumlycopersicum [M. incognita is limited, in contrast to recent advances in other plants. As M. incognita invasion is initially sensed by tobacco roots; the root is the ideal tissue for determining the tobacco resistance response and adaptation to M. incognita.Comparative transcriptome analysis using RNA sequencing (RNA-Seq) has been widely used to investigate non-model and model species . Unlike vulgaris , Cucumisuliferus , and Solpersicum . HoweverM. incognita invasion. This study aimed to identify relevant genes and pathways associated with tobacco resistance and elucidate the complex regulatory networks associated with the response to M. incognita. In this study, tobacco was used as a model to identify the physiological and transcriptional changes in the resistant K326 and susceptible Changbohuang genotype in response to Two typical tobacco genotypes: non-inoculated K326 , inoculated K326 , Changbohuang , and Changbohuang  were used in this study. The results revealed significant difference in the biomass of nematode-inoculated and control plants. Line K326 was only slightly affected in comparison with Changbohuang . The relThe disease index (DI) was investigated in plants after 60 days of infection with RKNs, as described by Wang et al. . The aveM. incognita invasion, we measured activities of POD, polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL), as well as the content of malondialdehyde (MDA). No considerable differences in the four parameters were noted between RC and SC before M. incognita infection. However, the values remarkably increased in RI and SI samples after M. incognita infection | ≥ 1 and p < 0.05 as significant cutoff criteria. By using these criteria, we detected 4353 differentially expressed genes (DEGs) in the resistant genotype, of which 2540 were upregulated and 1813 were downregulated. We also detected 545 DEGs in the susceptible genotype, of which 188 were upregulated and 357 were downregulated  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed the biological processes, cellular components, molecular functions, and metabolic pathways associated with the identified transcripts from tobacco roots. The 291 putative resistance-related DEGs were divided into three principal GO categories: biological process, cellular component, and molecular function . Sixteenp-value < 0.05), including “phenylpropanoid biosynthesis”, “tropane, piperidine, and pyridine alkaloid biosynthesis”, “tyrosine metabolism”, “isoquinoline alkaloid biosynthesis”, “pentose and glucuronate interconversions”, and “starch and sucrose metabolism”  are known to be the key antioxidant enzymes that regulate plant extracellular H2 levels . In thisenotypes .PR1 was upregulated, with 2.5594- and 1.2235-fold increases in the resistant and susceptible genotype, respectively , which was upregulated in the resistant genotype but repressed in the susceptible genotype , was idehttp://plntfdb.bio.uni-potsdam.de/v3.0/). A total of seven DEGs related to nematode resistance in tobacco have been annotated and classified into four TF families (ethylene response factor (ERF), myoglobin (MYB), basic helix-loop-helix (bHLH), and indole acetic acid–leucine-resistant (ILR); MYB gene (gene_24648) in the RI samples was 10-fold higher than that in the RC samples  analysis . As expeM. incognita-resistant and susceptible genotypes, respectively. An average of 35 million clean reads were obtained, and approximately 82% of the sequences were successfully mapped to the reference genome  suggest that they play a crucial role in nematode–plant interactions . Hewezi M. incognita infection. Furthermore, the expression of 16 PRX genes varied markedly between the two genotypes  in a greenhouse. Egg masses were isolated from triturated tomato roots by using 0.5% NaOCl and then hatched at 26 °C. Freshly hatched J2s were preserved in deionized water. The experiment was conducted in a growth chamber under controlled conditions (16/8-h light/dark cycle at 26 °C and photosynthetic photon flux density of 350 μmol·m−2·s−1). The roots of 15 seedlings at a 10-leaf stage were inoculated with approximately 1000 J2s in 5 mL of deionized water, while control seedlings were mock-inoculated with the same volume of deionized water. A randomized complete block design was used in this experiment with three replicates.M. incognita- and mock-inoculated plants of K326 and Changbohuang. Samples were collected from three plants each. The POD activity was measured as described by Rao et al. [M. incognita, as described by Wang et al. [Root samples were harvested at 7 days post-inoculation from both o et al. ; PPO ando et al. . The MDAo et al. . At 60 dg et al. .Histopathological observation studies were conducted to determine the root cellular response in resistant and susceptible tobacco. The root tips were harvested from two genotypes at 7 d post inoculation. The roots were dehydrated with a graded ethanol series  and embedded in 58 °C-melting-point paraffin. Serial sections of thickness 10 μm were cut using a rotary microtome , and then deparaffinized and hydrated with water . The sections were stained for 2–5 min in toluidine solution, washed with water, observed under a microscope , differentiated based on color, and dried in an oven .The roots were thoroughly rinsed in tap water at 7 days post-inoculation and then immediately frozen in liquid nitrogen until RNA extraction. The treatments were as follows: non-inoculated K326 (RC), inoculated K326 (RI), non-inoculated Changbohuang (SC), and inoculated Changbohuang (SI). Total root RNA was extracted using the TRIzol reagent , and then four sequencing libraries were constructed using the NEBNext Ultra directional RNA library prep kit for Illumina , in accordance with the manufacturer’s protocols. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2000 platform, and 150-bp paired-end reads were generated.https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.0/), and clean reads were aligned to the reference genome (ftb://anonymous@ftp.solgenomics.net/genomics/Nicotiana_tabacum/assembly/K326) using TopHat v2.0.12 (http://tophat.cbcb.umd.edu/). We selected TopHat as the mapping tool because it can generate a database of splice junctions based on the gene model annotation file and thus produces better mapping results than other, non-splice mapping tools do.Raw reads in the FASTQ format were processed using Perl scripts. In this step, clean reads were acquired by removing reads containing an adapter, low-quality reads, and reads containing poly N. At the same time, Q20, Q30, and the GC content were calculated for the clean data. All subsequent analyses were based on high-quality clean reads. The reference genome and gene annotation files were downloaded directly from the genome website. An index of the reference genome was built using Bowtie v2.2.3 (http://bioinfo.au.tsinghua.edu.cn/software/degseq) was used to analyze the differential expression between two samples. The false discovery rate was controlled by adjusting the p-values by using the Benjamini and Hochberg method [p-value of < 0.05 and the absolute value of the log2 ratio of ≥1 were set to evaluate the significance of DEGs. GO annotation of DEGs was performed using the GO seq v2.12 (http://bioconductor.org/packages/goseq/). Next, the KEGG enrichment analysis was implemented using the KOBAS software v2.0 . Clean reads were aligned to the K326 tobacco genome using the TopHat v2.0.12 software. A fragments per kilobase per million (FPKM) analysis, which simultaneously considers the sequencing depth and length for this count , was useg method . Thresho−ΔΔCt method was used to calculate fold changes in gene expression [Fifteen DEGs were selected to test the reliability of the RNA-Seq data by qRT-PCR with three biological replicates. Gene-specific primers were designed using Primer Premier 5.0 see  and syntpression .


Funneliformis mosseae IMA1 and the wheat root endophytic bacterium Lactobacillus plantarum B.MD.R.A2. These microorganisms were inoculated, alone or in combination, in durum wheat (cultivars Odisseo and Saragolla). Non-inoculated plants of each cultivar represented the controls. Forty-three days after sowing, roots were deprived of the epiphytic microbiota and subjected to DNA extraction. The DNA was used as template in PCR-DGGE analysis of the 16S rRNA gene (variable region V3–V5) and 16S (region V1–V3) metagenetics. Odisseo and Saragolla root endophytic bacterial biotas differed for number of OTUs and composition. In detail, Pseudomonas was higher in Odisseo than in Saragolla. The inoculation of F. mosseae and L. plantarum increased the abundance of Pseudomonas, some Actinobacteria  strains) and Bacteroidetes in both cultivars. However, the endophytic bacterial biota of Saragolla roots inoculated just with lactobacilli did not differ from that of the control. The inoculation of Saragolla with F. mosseae, alone or in combination with lactobacilli, led to higher abundance of Rhodococcus, belonging to Actinobacteria and encompassing PGP strains. First, this work showed that F. mosseae and L. plantarum shape the endophytic bacterial biota of durum wheat roots. Abundance of some OTUs was affected by the microbial inoculation, depending on the cultivar. This result represents a starting point for exploitation of beneficial endophytes of wheat roots.This study aimed at characterising the endophytic bacterial communities living in durum wheat roots, as affected by wheat cultivar and inoculation of the Arbuscular mycorrhizal fungus  Glomeromycota) and their associated bacteria, which positively interact with plant roots , AMF transfer soil mineral nutrients, such as phosphorus (P), nitrogen (N), sulfur (S), potassium (K), calcium (Ca), copper (Cu), and zinc (Zn), from the soil to the host plant, by means of a fine network of extraradical hyphae which grow from mycorrhizal roots into the surrounding soil . Beyond Cellvibrio, Chondromyces, Flexibacter, Lysobacter, Pseudomonas (Arthrobacter), Bacillales , Rhizobiales , Pseudomonadales , Burkholderiales , Streptococcus and Ensifer  endophytes may colonise or not plant tissues, depending on different variables and environmental conditions , one of the most important food crops worldwide, has been studied for its susceptibility to mycorrhizal colonisation, with 108 accessions characterised by the identification of genetic markers associated to such a trait  gene and 16S metagenetics through Illumina MiSeq platform. Our data provide knowledge of different factors shaping the composition of endophytic bacterial communities of durum wheat roots, opening the way to the exploitation of beneficial plant endophytes in sustainable management strategies.Lactic acid bacteria (LAB), such as , longan  and are , longan . LAB hav, longan . Recent fe cycle . Durum w a trait . NothingF. mosseae (T. H. Nicolson and Gerd.) C. Walker and A. Schüssler (formerly known as Glomus mosseae), maintained in the collection of Microbiology Labs  of the Department of Agriculture, Food and Environment, University of Pisa, Italy (isolate number IMA1). The holotype is deposited in the herbarium of the Department of Biology, University of Pisa, Herbarium Horti Botanici Pisani as PI-HM-Z4. The inoculation was obtained from Medicago sativa L. pot cultures in a mixture of sandy loam soil and calcinated clay  (1:1 v/v) which was steam-sterilised (121°C for 30 min on two consecutive days) to kill naturally occurring AMF. Chemical and physical characteristics of the soil used were as follows: pH (H2O), 8.0; clay, 15.3%; silt, 30.2%; sand, 54.5%; organic matter, 2.2% ; total N, 1.1 g kg–1 (Kjeldahl); extractable P, 17.6 mg kg–1 (Olsen). Sterile seeds of M. sativa L. were sown, and plants grown for 4 months, then shoots were excised and roots chopped into fragments. Then the substrate was air dried at room temperature and utilised as crude inoculation. The activity of such inoculum was verified by a mycorrhizal inoculum potential (MIP) bioassay  washed twice in sterile physiological saline solution (0.9% NaCl) and finally suspended in physiological solution, as described in 6 CFU g–1.la roots , was culla roots  at 28°C F. mosseae crude inoculation with sterile calcinated clay. Control and bacterial treatments received a mock inoculation produced by sterilising the appropriate amount of mycorrhizal inoculation. All tubes received 2 mL of a filtrate, obtained by sieving the mycorrhizal inoculation through a 50-μm pore diameter sieve and a Whatman paper no. 1 , to ensure common microbiota for all treatments. L. plantarum B.MD.R.A2 was inoculated at sowing of durum wheat (Triticum turgidum ssp. durum) seeds and 15 days after germination using 1 mL of the bacterial suspension per tube, while the same amount of physiological saline solution was provided to treatments not inoculated with lactobacilli.The experiment consisted of four treatment groups: control, AMF inoculum (M), bacterial inoculum (B), AMF and bacterial inoculum (BM). The microcosm experiment was carried out using 50 mL Falcon tubes. The growth substrate was prepared mixing (1:1 v/v) the Before microbial inoculation, durum wheat seeds of the cultivars Odisseo (O) and Saragolla (S) were surface sterilised twice in 1.5% (v/v) sodium hypochlorite solution for 10 min and thoroughly rinsed with sterile MilliQ water. Three seeds were sown in each tube, which was put in sun-transparent bags and maintained in a growth chamber at 27°C under a 16/8 h light/dark daily cycle until harvest. Plantlets were thinned to one per tube at emergence. Six replicates were performed per each treatment. The plantlets were watered as needed. Forty-three days after sowing, plants were harvested, roots removed from soil and washed with tap water. Three replicate plants were used for assessing mycorrhizal colonisation and the other three for extraction of total DNA. In order to measure the level of mycorrhizal colonisation, roots were cleared in 10% (w/v) KOH in a 80°C water bath for 15 min and stained with Trypan blue in lactic acid (0.05%) after 10 min in 2% aqueous HCl. The percentage of AMF colonisation was calculated using a dissecting microscope at ×25 or ×40 magnification and the gridline intersect method .Before DNA extraction, 10 g of plant roots were washed and surface sterilised as described in For the analysis of endophytic bacterial communities, the amplification of the variable region V3–V5 of 16S ribosomal DNA (rDNA) was carried out using the primers 341F (CCTA CGGGAGGCAGCAG) and 907R (CCGTCAATTCCTTTRAG TTT) . At its TM Universal Mutation Detection System . A composite mix of bacterial 16S rRNA gene fragments from L. plantarum IMA B23, L. brevis DSMZ 20054, Pseudomonas fluorescens 19/5, Streptomyces sp. IMA AC3, Ensifer meliloti IMA TSA26, Fictibacillus barbaricus IMA TSA50, Streptomyces sp. IMA W64 were added as reference DGGE marker (M). Gels were run and visualised as described in For the DGGE analysis, amplicons were separated in 8% (w/v) polyacrylamide gels with a 30–70% urea-formamide gradient, using the DCodePCR was performed at RTL Genomics , using primers 28F/519R, targeting a region spanning from V1 to V3 of Bacteria . The Ill1. The related sequences were collected and aligned using MUSCLE  using Statistica 7.0 for Windows.DGGE profiles were digitally processed with BioNumerics software version 7.6  as reported in g MUSCLE , b, and g MUSCLE  in Mega LR536406 to LR536425. The 16S rRNA gene sequences obtained from 16S metagenetic analysis are available in the Sequence Read Archive of NCBI (accession number PRJNA543396).The sequences obtained by PCR-DGGE bands were submitted to the European Nucleotide Archive under the accession numbers from Funneliformis mosseae successfully established mutualistic symbiosis with durum wheat roots, with percentages of colonised root length ranging from 8% to 27%, with no differences between the two cultivars Odisseo and Saragolla. No colonisation was observed in non-inoculated plants.DNA fragments of approximately 600 bp, corresponding to the V3–V5 region of the 16S rRNA gene, were successfully amplified from all samples. DGGE analyses of PCR products showed complex profiles characterised by intense and clearly defined fragments . The endThe cluster corresponding to the cultivar Saragolla was further split into two sub-clusters (77% similarity). The first one contained samples inoculated with AMF (S-M) and co-inoculated with AMF and LAB (S-BM) showing a similarity of 81%, while the second one grouped control (S) and samples inoculated with LAB (S-B), at a similarity of 89%.R = 0.7448, p < 0.05).The differences observed between the endophytic community composition associated with the two durum wheat cultivars were also confirmed by NMDS analysis of the DGGE profiles . ANOSIM Biodiversity indices of PCR-DGGE profiles did not differ among the treatment groups (data not shown).Massilia tieshanensis, Massilia aurea, Methylibium, and Hydrogenophaga intermedia), Firmicutes (Tumebacillus ginsengisoli), Actinobacteria (Rhodococcus), and Bacteroidetes   , 4.N. populi, F. kyonggii, M. aurea, M. tieshanensis, H. intermedia, T. ginsengisoli, and Rhodococcus. The inoculated samples were characterised by higher and lower intensity bands corresponding to O. koreensis and Massilia and T. ginsengisoli, respectively, compared with the non-inoculated control.In particular, the main endophytic species characterising control Odisseo roots were represented by T. ginsengisoli. Moreover, in Saragolla control N. populi and Rhodococcus were less abundant. In the inoculated samples, with the exception of S-B, N. populi, O. koreensis, Methylibium, and Rhodococcus were well represented, while the bands corresponding to F. kyonggii, H. intermedia, and Massilia species were less intense  strongly differed between Odisseo (35.8%) and Saragolla (ca. 2.8%) roots (F. mosseae or the LAB L. plantarum increased the relative abundance of Pseudomonadaceae in both cultivars. The co-inoculation of both F. mosseae and L. plantarum resulted in a further increase of Pseudomonadaceae (ca. 77.2%) only in Odisseo roots.Total DNA from the endophytic fraction of wheat roots was used as template to amplify the V1–V3 region of 16S rRNA gene. Amplicons were subjected to high throughput sequencing on the Illumina MiSeq platform. A total of 264,504 quality-trimmed sequences of 16S rRNA gene amplicons were obtained, with an average of 33,063 sequences/sample, and an average length of 509 bp (data not shown). The Good’s Estimated Sample Coverage was higher than 99.9% for all the samples . Controlisseo 35.% and Sar%) roots . The inoRhizobium. The inoculation of L. plantarum and/or F. mosseae resulted in a decrease of the relative abundance of Rhizobiaceae. Hyphomicrobiaceae (mainly Devosia), Oxalobacteraceae (Duganella and Massilia genera) and Enterobacteriaceae (Pantoea) were the other dominant Proteobacteria in Saragolla roots. These families decreased in roots inoculated with L. plantarum and/or F. mosseae. The only exception was Pantoea agglomerans, which was found at the highest level in Saragolla roots inoculated with F. mosseae , found in the same relative amount in the non-inoculated roots (ca. 4.5%), decreased in inoculated Odisseo roots and increased in L. plantarum and/or F. mosseae inoculated Saragolla roots. Saragolla roots harboured higher Sphingomonadaceae than Odisseo roots. The relative abundance of some species belonging to Sphingomonadaceae  was the highest in roots of Saragolla inoculated with F. mosseae (Cellvibrio genus) was detected only in L. plantarum and/or F. mosseae inoculated Odisseo and Saragolla roots. On the contrary, Burkholderiaceae  disappeared in Odisseo and Saragolla roots co-inoculated with L. plantarum and F. mosseae. Comamonadaceae, including Hydrogenophaga intermedia, was found at the highest level in non-inoculated Odisseo and Saragolla roots.Rhizobiaceae harboured higher relative abundance in Saragolla than in Odisseo roots . The main OTU component of Rhizobiaceae was the genus  mosseae . Sub-dom mosseae . CellvibRhodococcus genus) strongly differed between Odisseo (12%) and Saragolla (ca. 4.7%) roots. The inoculation of roots with L. plantarum and/or F. mosseae led to a decrease of Rhodococcus species (R. erythropolis and Rhodococcus sp.) in Odisseo. Opposite effect was found in the inoculated Saragolla roots. Other Actinobacteria families were variously detected at sub-dominant level among samples. Within them, Microbacteriaceae (Microbacterium), Micromonosporaceae (Actinoplanes), and Streptomycetaceae (Streptomyces europaeiscabiei) increased in inoculated Odisseo and Saragolla roots. Opposite trend was found for Micrococcaceae (Arthrobacter).Within Actinobacteria, the relative abundance of Nocardiaceae (mainly Ohtaekwangia) which was found at the highest level in Saragolla roots inoculated with L. plantarum and F. mosseae, as revealed also by PCR-DGGE sequencing (Chryseobacterium) was detected only in inoculated samples. Chitinophagaceae and Sphingobacteriaceae decreased in inoculated Odisseo roots. In Saragolla roots similar trend was observed for Sphingobacteriaceae. On the contrary, Chitinophagaceae, with the genus Niastella, increased in F. mosseae and, especially, in Saragolla roots co-inoculated with AMF and LAB, confirming PCR-DGGE findings  than Saragolla, which showed a high diversity, without dominant taxa. Such results complement and support previous data on the composition of rhizosphere bacteria of wheat, varying with genotype  on wheat plants enhanced phosphate solubilisation, indolacetic acid production, rate of seed germination and plant biomass , while the abundance of other genera, such as Streptomyces and Microbacterium spp., was enhanced in both cultivars. Actinobacteria, frequently isolated from the soil, are considered key soil beneficial bacterial taxa, being able to produce many different biologically active secondary metabolites, including antibiotics, anticancer and immunosuppressant drugs, and degrading organic matter enzymes  was the highest in Saragolla wheat roots inoculated with F. mosseae. The N-fixing functional trait of Sphingomonas spp. is of particular interest, suggesting their possible role in the promotion of plant growth. Actually, the inoculation of wheat seeds with a strain of Sphingomonas sp. increased root biomass accumulation and the concentration of nutrients , which are widely utilised as biofertilizers, and/or LAB (L. plantarum) represent significant ecological drivers in the establishment of the bacterial endophytic community in durum wheat roots. The increase of several bacterial species and genera encompassing PGP strains, promoted by wheat inoculation of LAB and, especially, AMF, represents a starting point for the exploitation of beneficial endophytes of wheat roots. Further studies are necessary to confirm the results shown in this paper and to better understand the dynamics of endophytes establishment in wheat roots, as modulated by the complex interactions among AMF, mycorrhizosphere bacteria and wheat roots.In conclusion, our work for the first time showed that AMF (The datasets generated for this study can be found in the Sequence Read Archive of NCBI, PRJNA543396.MA coordinated plant and PCR-DGGE experimental activities and wrote the manuscript. MP performed the PCR-DGGE and statistical analyses on results from PCR-DGGE. CC performed the experiments with microorganisms inoculated in plants. NC extracted DNA and elaborated the results from 16S metagenetics. MGi conceived the study and wrote the manuscript. MD revised the manuscript. MGo conceived the study. FM coordinated the 16S metagenetics experimental activities and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wound infections are responsible for significant human morbidity and mortality worldwide. Specifically, surgical site infections are the third most commonly reported nosocomial infections accounting approximately a quarter of such infections. This systematic review and meta-analysis is, therefore, aimed to determine microbial profiles cultured from wound samples and their antimicrobial resistance patterns in Ethiopia.Literature search was carried out through visiting electronic databases and indexing services including PubMed, MEDLINE, EMBASE, CINAHL, and Google Scholar. Original records, available online from 2000 to 2018, addressing the research question and written in English were identified and screened. The relevant data were extracted from included studies using a format prepared in Microsoft Excel and exported to STATA 15.0 software for analyses of outcome measures and subgrouping. Der-Simonian-Laird’s random effects model was applied for pooled estimation of outcome measures at 95% confidence level. Comprehensive meta-analysis version-3 software was used for assessing publication bias across studies. The study protocol is registered on PROSPERO with reference number ID: CRD42019117638.S. aureus was 36% , from which 49% were methicillin resistant strains. The pooled estimate of E. coli isolates was about 13%  followed by P. aeruginosa, 9% , K. pneumoniae, 9%  and P. mirabilis, 8% . Compared to other antimicrobials, S. aureus has showed lower estimates of resistance against ciprofloxacin, 12%  and gentamicin, 13% . E. coli isolates exhibited the highest point estimate of resistance towards ampicillin . Gentamicin and ciprofloxacin showed relatively lower estimates of resistance with pooled prevalence being 24%  and 27% , respectively. Likewise, P. aeruginosa showed the lowest pooled estimates of resistance against ciprofloxacin .A total of 21 studies with 4284 wound samples, 3012 positive wound cultures and 3598 bacterial isolates were included for systematic review and meta-analysis. The pooled culture positivity was found to be 70.0% . Regarding the bacterial isolates recovered, the pooled prevalence of S. aureus is by far the most common bacterial isolate recovered from wound infection. The high estimate of resistance was observed among β-lactam antibiotics in all bacterial isolates. Ciprofloxacin and gentamicin were relatively effective in treating wound infections with poly-microbial etiology.Generally, the wound culture positivity was found very high indicating the likelihood of poly-microbial contamination. The online version of this article (10.1186/s40360-019-0315-9) contains supplementary material, which is available to authorized users. Wound provides a warm, moist, and nutritive environment which is conducive to microbial colonization, proliferation, and infection. It can occur during trauma, accident, burn, surgical procedures or as a result of chronic disease conditions such as diabetes mellitus and leprosy , 2. All Staphylococcus aureus and Coagulase Negative Staphylococci (CoNS). Besides, gram-negative aerobic bacilli such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis are the most prevailing clinically relevant isolates [S. aureus particularity Methicillin resistant strains (MRSA) as well as P. aeruginosa are typical biofilm producers making the wound infection difficult to treat using standard antibiotics [Though it varies based on the wound source, the most commonly isolated gram-positive cocci are isolates –9. Amongibiotics , 10–12.Treatment of wound infections with antimicrobial agents as well as optimum treatment regimens remains ill defined. Many published guidelines are mainly based on expert opinion rather than evidence-based data. The selection of appropriate antimicrobial agents has been inconclusive. Though prophylactic use of antimicrobials can help reduce the risk of infection and promotes wound healing, it is not a direct substitute for good local wound care such as irrigation and surgical debridement. Moreover, judicious use of antimicrobials reduces the development of antimicrobial resistance (AMR) –15. Revihttp://www.crd.york.ac.uk/PROSPERO/display_record.php?ID=CRD42019117638The identification and screening of studies as well as eligibility assessment of full texts was conducted in accordance with the preferred reporting items for systematic review and meta-analysis (PRISMA) statement . In addiLiterature search was carried out through visiting electronic databases and indexing services. The PubMed, MEDLINE (Ovid®), EMBASE (Ovid®), CINAHL (EBSCOhost), and Google Scholar, were used as main source of data. Besides, other supplementary sources including ResearchGate, WorldCat, Science Direct and University repositories were searched to retrieve relevant data. Excluding the non-explanatory terms, the search strategy included important keywords and indexing terms: wound, wound infection (MeSH), “antimicrobial susceptibility”, bacteria (MeSH), “antimicrobial resistance”, “antibacterial resistance”, and “Ethiopia”. The MeSH terms, Boolean operators , and truncation were applied for appropriate searching and identification of records for the research question.Studies conducted in EthiopiaStudies available online from 2000 to 2018Studies published in English languageLaboratory-based observational  studies addressing the research question: studies dealing with wound culture positivity and prevalence of individual isolates recovered from wound samples as primary outcome measuresAny review papers published in the study periodStudies having mixed sample sources Wound samples taken from animalsIrretrievable full texts (after requesting full texts from the corresponding authors via email and/or Research Gate account)Studies with unrelated or insufficient outcome measuresStudies with outcomes of interest are missing or vagueAlong with application of appropriate limits, online records from each database or directory were exported to ENDNOTE reference software version 8.2 . The records were then merged to one folder to identify and remove duplicates with the help of ENDNOTE and/or manual tracing as there is a possibility of several citation styles per study. Thereafter, two authors Mekonnen Sisay (MS) and Dumessa Edessa (DE) independently screened the title and abstracts with the predefined inclusion/exclusion criteria. Records which passed the screening phase were subjected for eligibility assessment of full texts. For this, two authors, MS and Teshager Worku (TW) independently collected full texts and evaluated their eligibility for meta-analysis. In each case, the third author was consulted to solve disagreement occurred between the two authors.Using standardized data abstraction format prepared in Microsoft Excel , at University of Adelaide, Australia . The purThe primary outcome measures are the culture positivity of wound samples and the prevalence of bacterial isolates recovered from infected wound samples in Ethiopia. The secondary outcome measure is the antimicrobial resistance profiles of clinically relevant bacterial isolates against commonly prescribed antimicrobial agents taken from different pharmacologic categories . Subgroup analysis was also conducted based on the nature of wound sources .2 statistics. Forest plots and summary tables were used to report the results. Comprehensive Meta-analysis software version-3, , was employed for assessment of publication bias. The presence of publication bias was evaluated by using Egger’s regression and Begg’s and Mazumdar’s correlation tests and presented with funnel plot. All statistical tests with p-values less than 0.05 (one-tailed) were considered significant [The extracted data were imported from Microsoft Excel to STATA software, version 15.0 for the pooled estimation of outcome measures. Sensitivity and subgroup analyses were also conducted to minimize the degree of heterogeneity. Der-Simonian-Laird’s random effects model was applied for the analysis at 95% confidence level. Heterogeneity among the included studies was assessed with Inificant , 23.A literature search in electronic databases including PubMed, MEDLINE, EMBASE, CINAHL and Google Scholar retrieved a total of 111 studies. From which, 62 studies were found duplicate through ENDNOTE and manual tracing. The remaining studies were screened using their titles and abstracts and 21 of them did not fulfill the inclusion criteria and thus removed from further eligibility study. The full texts of 28 studies were thoroughly assessed to ensure the presence of at least the primary outcome measures in sufficient and non-ambiguous way. In this regard, 7 studies did not meet the inclusion criteria and thus removed from final inclusion. Therefore, 21 studies addressing the outcome of interest were included  and gram-negative  bacterial isolates recovered from positive cultures of wound samples  were summarized against commonly prescribed antimicrobial agents taken from various pharmacologic classes: penicillins , cephalosporins (ceftriaxone), fluoroquinolones (ciprofloxacin), marcrolides (erythromycin), sulfonamides (cotrimoxazole) and aminoglycosides (gentamicin). Unlike others, a study conducted by Hailu et al. utilized variable number of bacterial isolates per antimicrobial agent . AntimicIn this meta-analysis, a total of 3012 positive bacterial cultures obtained from 4284 wound samples were included. The pooled prevalence of culture positive cases from all wound samples was found to be 70.0%        Fig. , K. pneu1%) Fig.  and P. mS. aureus isolates, the prevalence of Methicillin resistant S. aureus (MRSA) strains was found to be 49% . The combination of amoxicillin with β-lactamase inhibitor clavulanic acid (Coamox-clav) reduced the amoxicillin resistance by 42% . Besides, the pooled estimates of amoxicillin and ampicillin resistance among CoNS isolates were 62%  and 72% , respectively. Among all antimicrobial agents tested, S. aureus exhibited relatively lower estimates of resistance against ciprofloxacin, 12%  and gentamicin, 13% . Likewise, ciprofloxacin had the least resistant isolates  followed by gentamicin  for CoNS. The 3rd generation cephalosporin ceftriaxone resistance was observed among gram-positive isolates with estimates being 36%  and 37%  for S. aureus and CoNS, respectively  followed by amoxicillin . Gentamicin and ciprofloxacin showed relatively lower estimates of resistance with pooled prevalence being 24%  and 27% , respectively. Amoxicillin-clavulanic acid combination reduced the amoxicillin resistance of E. coli by 16% . Nearly half  and more than half  of E. coli isolates were found resistant to ceftriaxone and cotrimoxazole, respectively in wound samples in Ethiopia . Only 10% reduction in overall resistance was observed in amoxicillin-clavulanate  compared to amoxicillin alone  in P. aeruginosa isolates.. More than half of P. aeruginosa isolates  developed resistance to ceftriaxone. Moreover, around three-fourths of these isolates  were found resistant to cotrimoxazole  were also tested against seven antimicrobial agents. The antimicrobials with highest estimates of resistance were found to be amoxicillin and ampicillin in both bacterial isolates. Ciprofloxacin followed by gentamicin were antimicrobials having lower point estimates of resistance in both types of isolates. More than half of K. pneumoniae isolates exhibited ceftriaxone resistance    Fig. .Fig. 10FS. aureus with pooled estimate being 36% which is by far higher than the prevalence of CoNS, E. coli, P. aeruginosa, K. pneumoniae and other gram-negative aerobic bacilli. This is further supported by several published studies which reported that gram-positive aerobic cocci, particularly, Staphylococci, have been the primary cause of wound infections [S. aureus has been a single most common pathogen isolated from several wound samples [E. coli, P. aeruginosa, K. pneumoniae, and P. mirabilis. This finding is in trajectory with review of recent literature in different settings [In this systematic review and meta-analysis, more than two-thirds of wound samples 70%) were found culture positive. The subgroup analysis has also indicated higher culture positivity of burn and other non-surgical wounds compared to surgical wounds regardless of prophylactic antibiotic status. In concordant with this, a five year study conducted by Agnihotri et al reported high culture positivity 96%) from burn wound infections % from bu. The low0% were ffections –50. More samples –49, 51. settings , 48, 52.S. aureus and P. aeruginosa [S. aureus was the most prevalent in the first week of wound infection with an overall prevalence of about 28.4%. It was, however, surpassed by P. aeruginosa from the third week onwards [S. aureus and P. aeruginosa are opportunistic pathogenic bacteria and are widely known to cause chronic biofilm-based wound infections [Though it seems controversial, many studies indicated that the two highly prevalent bacterial isolates recovered from chronic wound infections were ruginosa , 53, 54. onwards . On the fections . These pfections .S. aureus isolates were characterized as MRSA strains. This is a clear direction of how high the healthcare settings become a reservoir of resistant strains like MRSA. Likewise, a summary of wound infections in orthopedics indicated that S. aureus constituted about 34% of all isolates and 48% of this isolate were reported to be MRSA [The overall prevalence of MRSA strains was found to be 49% indicating one in every two  be MRSA . Conditi be MRSA , 57., S. aureus has developed resistance to majority of β-lactam antibiotics . As the analysis tried to address, the third generation cephalosporin ceftriaxone resistance was estimated to be 36%. Though it is less pathogenic than S. aureus, CoNS had also developed closer estimates of resistance in these antibiotics. Most notably, the steady erosion of the effectiveness of β-lactam antibiotics for treatment of S. aureus infection is due to the fact that it’s extraordinary adaptability in developing resistance with several mechanisms [Staphylococci such as S. aureus produce β-lactamase enzymes which in turn degrade the β-lactam ring of majority of β-lactam antibiotics  and make these drugs devoid of any antibacterial activity. Though they have a wide range of bacterial coverage, the β-lactamase sensitivity of broad (ampicillin and amoxicillin) and extended spectrum  penicillins are no longer effective for β-lactamase producing Staphylococci such as S. aureus [S. aureus showed high estimate of resistance (about 75%) to standard β-lactam antibiotics whereas coamox-clav resistance was reduced to 23.5% [Due to β- lactamase producing capability and modification of penicillin binding proteinschanisms , 56, 57.. aureus , 56, 58.S. aureus has also developed resistant to β-lactamase resistant penicillins  with apparently different mechanism. From experience, resistant to these agents was historically treated as MRSA since the first agent in this class of penicillins is methicillin [S. aureus acquires resistance genes which encode a modified form of penicillin binding proteins (PBP). In MRSA, the horizontally acquired mecA gene encodes PBP2A. PBP2 is the only bifunctional PBP in S. aureus. In MRSA strains, the essential function of PBP2 may be replaced by PBP2A which functions as a surrogate transpeptidase. Since almost all β-lactams have little or no affinity to this protein, cross resistance occurs regardless of β-lactamase stability status [S. aureus strains were found resistant to ceftriaxone. Apart from this, macrolides (erythromycin) and sulfa drugs (cotrimoxazole) resistance was observed in about one-thirds of S. aureus strains. Generally, this study showed that more than 10% of S. aureus isolates were also resistance to ciprofloxacin and gentamicin. CoNS had also showed higher gentamicin resistance than S. aureus.Moreover, hicillin –61. S. ay status , 60. ThiP. aeruginosa was about 77%. Likewise, more than 50% of P. aeruginosa and K. pneumoniae isolates had developed resistance to the third generation cephalosporin ceftriaxone. From sulfa drug combination, cotrimoxazole resistance was observed in more than half of all isolates in each gram-negative bacterium with the highest resistance estimate (76%) observed in P. aeruginosa. Even if the degree of susceptibility varies across strains, relatively lower estimates of resistance were observed in ciprofloxacin and gentamicin making these agents relatively effective for treatment of wound infections.Observing the AMR profile of gram-negative bacteria, a high estimate of resistance was observed in β-lactam antibiotics. All gram-negative bacilli have developed clinically significant resistant against broad spectrum penicillins (amoxicillin and ampicillin). The combination of β-lactamase inhibitor clavulanic acid with amoxicillin did not show an appreciable reduction. The Coamox-clav resistance in Enterobacteriaceae [K. pneumoniae and E. coli isolated from wound samples, around 11% produced ESBL  [In this regard, a plasmid encoded extended-spectrum β-lactamases (ESBLs) production has been alarmingly increasing in gram-negative bacteria particularly in eriaceae –64. A stctively) . ESBLs hctively) , 65–67.P. aeruginosa has naturally known to develop resistance by its outer membrane (porine channels) permeability problems coupled to adaptive mechanisms such as efflux pumps and can readily achieve clinical resistance. Almost all penicillins, except the fourth generation series , are not clinically effective at all for the treatment of Pseudomonas infections.. Likewise, majority of the third generation cephalosporins and all of the prior generations are no longer effective since they are not able to penetrate the porine channels of this bacterium though other resistance mechanisms should also be considered. Generally, this bacterium is highly notorious to develop resistance to multiple antibiotics and has joined the ranks of ‘superbugs’ due to its enormous capacity to engender resistance [The difficult to treat gram-negative bacteria sistance , 68–73.S. aureus and P. aeruginosa are among the pathogen of interest and have several resistance mechanisms making the treatment challenging. In general, the most common drugs used to treat S. aureus infections include amoxicillin/clavulanate, ampicillin/sulbactam, and cloxacillin whereas MRSA strains are better treated with levofloxacin, vancomycin, linezolid and tigecycline. In the presence of co-infections related to S. aureus and P. aeruginosa, piperacillin/tazobactam, carbapenems and ciprofloxacin may represent the first choice for treatment of wound infections [Historically, the classes of antibiotics used in the treatment of wound infections include the β -lactams and aminoglycosides . At presfections .Staphylococci have been the predominant gram-positive cocci in wound infection with S. aureus being by far the most prevalent isolate. This finding alarms the high load of MRSA strains in hospital settings as well. Gram-negative aerobic bacilli under Enterobacteriaceae are also commonly isolated pathogens in wound infections. Though it is expected in S. aureus and P. aeruginosa, all isolates exhibited highest estimates of resistance against β-lactam antibiotics. Most notably, resistance to the 3rd generation cephalosporin ceftriaxone was observed in more than 40% of all gram-negative isolates . Though more than 10% of resistance was reported in all isolates, ciprofloxacin and gentamicin were relatively good in treating wound infections with poly-microbial etiology. Hence, considering microbial epidemiology and AMR patterns, treatment should be tailored for addressing poly-microbial etiology thereby effectively manage wound infection and its complication as well as preserve antimicrobials and contain AMR.Generally, the wound culture positivity was very high indicating the likelihood of poly-microbial load. Surgical wounds had relatively lower culture positivity than non-surgical counterparts though the role of prior local wound care following surgery is yet to be investigated. Even if the study has extensively included all relevant data regarding wound infection, it was not without limitations. There was no clear demarcation of wound samples in inpatient and outpatient settings. Besides, there was no documented history of prophylactic use of antibiotics in individual studies though we conducted subgroup analyses to reduce culture positivity/negativity errors in surgical patients. The antimicrobial susceptibility testing was a little bit different across studies. Therefore, this meta-analysis should be seen in the context of such limitations.Additional file 1:Table S1. Completed PRISMA checklist: The checklist highlights the important components addressed while conducting systematic review and meta-analysis from observational studies (DOC 66 kb)Additional file 2:Table S2. Data abstraction format with crude data: The table presented the ways of data collection (study characteristics and outcome measures) in Microsoft excel format. (XLSX 38 kb)


Strongyloides stercoralis and HIV have been reported to coexist, particularly in resource-limited settings such as India. In an immunocompromised host, S. stercoralis can progress to strongyloidiasis hyperinfection syndrome (SHS). However, SHS is not common in patients with advanced HIV disease. Immune reconstitution inflammatory syndrome (IRIS) developing after initiation of antiretroviral therapy (ART) can target multiple pathogens including S. stercoralis. The authors present here a 46-year-old HIV-infected female who was recently diagnosed with HIV-1 infection, started ART, and developed SHS. Her upper GI endoscopy revealed severe gastroduodenitis, and X-ray chest showed extensive bilateral pneumonitis. We could identify S. stercoralis in induced sputum and duodenal biopsy. We could also identify gut inflammation to restrict invading parasites. After receiving antihelminthic therapy, she showed improvement, a course of events that fit the diagnosis of unmasking S. stercoralis IRIS.Parasitic infections such as  Strongyloides stercoralis typically infects the small bowel (duodenum and jejunum) of human hosts and is endemic to tropical and subtropical regions. The helminth enters the body through the skin and initially migrates through the venous peripheral blood system to the lung parenchyma. It ascends the tracheobronchial tree and is swallowed to reach the small intestine. After initial infection, mature females in the duodenum produce rhabditiform larvae that can develop into infective filariform larvae and reinfect the host through penetration of intestinal mucosa. This unique autoinfective capability facilitates chronic infection in a single host for decades [S. stercoralis remains a neglected tropical disease, and its association with HIV coinfection is sparsely studied in resource-limited settings such as India. It is reflected by the paucity of the published literature on HIV/S. stercoralis coinfection [S. stercoralis may be responsible for it. We hereby report a case of SHS as a component of IRIS in an HIV-positive individual recently initiated on ART in Pune, India. decades . Most im decades . S. sternfection –5. Sincenfection –12. ART-3, and plasma HIV-1 viral load was 867,000 copies/ml. She had no comorbidities or prior significant medical history. On examination, she was febrile (temperature: 100 degrees Fahrenheit) with pulse (100/min), blood pressure (110/60 mm Hg), and respiratory rate (24/min). Respiratory examination revealed crepitations in bilateral inframammary, infraaxillary, and infrascapular areas. There was diffuse abdominal tenderness but no organomegaly. Rest of the examination including fundoscopy was unremarkable. Hemoglobin was 8.3 g/dl while rest of the biochemical investigations was normal. CD4 count and plasma HIV-1 viral load after 1 month of ART was 190 cells/mm3 and 9,500 copies/ml, respectively, suggesting satisfactory immune reconstitution. The arterial blood gas (ABG) was suggestive of hypoxia (pO2-63 mm Hg) on room air. Chest X-ray was suggestive of bilateral, extensive, and patchy consolidation suggestive of infective etiology , oral steroids, and empirical antitubercular therapy (ATT) in addition to ART. She had diarrhea on admission, which subsided in 2 days after taking loperamide. Stool routine and modified Ziehl–Neelsen (ZN) examination was unremarkable. Due to a suspicion of CMV enterocolitis, she was also started on empirical oral valgancyclovir. Bronchoscopy with BAL was attempted but had to be aborted in view of worsening hypoxia during procedure. For establishing diagnosis, upper GI endoscopy and colonoscopy were attempted. Upper GI endoscopy revealed diffuse gastroduodenitis. Colonoscopy showed patchy colitis. Duodenal biopsy revealed focal blunting, ulceration, and neutrophilic exudates  and ivermectin (200 µg/kg). Inspite of antihelminthic treatment, her dyspnea worsened. Her repeat ABG showed hypoxia  and respiratory alkalosis. She developed hypotension (blood pressure 80/60 mm Hg) and altered sensorium in the form of increasing drowsiness . She was shifted to the Intensive Care Unit (ICU) for the same. MRI brain and CSF examination were normal while serum sodium level had dropped to 125 mEq/L. Noradrenaline infusion and noninvasive BIPAP ventilation were started. She was initiated on intravenous (i.v.) meropenem, i.v. teicoplanin, and i.v. fluconazole in addition to antihelminthic agents, steroids (in tapering doses), and ART. ATT and valganciclovir were withdrawn while TMP-SMX was reduced to prophylactic dose. She gradually started improving over the next 7 days with improvement in sensorium and reduction in breathlessness. She was shifted out of the ICU by day 21 of admission. Her CXR showed complete resolution of pneumonic consolidation. She was discharged on oral antihelminthic treatment which she continued for another 8 weeks. Follow-up endoscopy studies showed complete clearance of S. stercoralis.A 46-year-old female presented with complaints of fever, breathlessness on minimal exertion, vomiting, abdominal pain, and reduced appetite since 10 days. She also complained of weight loss of 10 kilograms in the last 6 months. She was diagnosed with HIV-1 infection, 1 month prior, and was prescribed tenofovir, lamivudine, and efavirenz fixed-dose combination single-pill regimen. Her baseline CD4 count was 68 cells/mmetiology . Sonograexudates . The surrcoralis . Lamina ory rate 4/min. ReS. stercoralis worldwide and the first from India. Advanced HIV has not been linked with disseminated strongyloidiasis despite a 21-fold increased risk of intestinal infection in endemic areas [S. stercoralis in HIV-infected patients ranged from 0% to 27.3% [+ cell counts, direct development of infective filariform larvae in the gut (autoinfection) is favored, compared with those with lesser immune function (low CD4 count or advanced HIV disease), in whom, indirect development is favored. Indirect development of filariform larvae in individuals with advanced HIV disease reduces the opportunity for dissemination to occur [S. stercoralis [S. stercoralis is listed as an immune reconstitution inflammatory syndrome- (IRIS-) related pathogen, this condition is scarcely reported in the literature [S. stercoralis, unmasking IRIS was seen in five cases [Our case report is the eighth reported case of IRIS to ic areas . Few preto 27.3% , 5. The to occur . T helpeto occur , 14. As to occur . This mircoralis –12. Two rcoralis . Althougve cases –10 whileve cases , 12. IRIS. stercoralis. We believe that the prevalence of this phenomenon is much higher in resource-limited settings such as India which is coendemic to HIV and S. stercoralis and has seen rapid scale up of ART in recent years. However, lack of awareness of health care personnel or lack of facilities in resource-limited settings to establish diagnoses leads to underreporting [In our patient, unmasking IRIS occurred 30 days after ART initiation and manifested as severe gastroduodenitis and patchy colitis leading to severe abdominal pain, vomiting, and intermittent diarrhea. In addition, pulmonary involvement was evidenced by dyspnea and bilateral pneumonitis on CXR leading to acute respiratory distress syndrome (ARDS). The presence of immune reconstitution was indicated by an increased CD4+ count and decreased viral load comparable to baseline. The temporal relationship between ART initiation and SHS in this case suggests a causal association between the two events. There are several possible explanations for this rapid clinical deterioration. First, a high parasite burden was present prior to ART, and the development of symptoms was a result of a renewed immune response to the pathogen that is typical of unmasking IRIS. Post-ART duodenal biopsy showed heavy parasite load coupled with moderate mononuclear infiltrate, eosinophil infiltration, and granuloma formation Figures  and 3. Seporting .In conclusion, physicians working in resource-limited settings such as India should consider parasitic infections as a differential diagnosis while evaluating cases of IRIS in HIV-infected patients starting ART.


The plant-specific Teosinte-branched 1/Cycloidea/Proliferating (TCP) transcription factor genes are involved in plants’ development, hormonal pathways, and stress response but their evolutionary history is uncertain. The genome-wide analysis performed here for 47 plant species revealed 535 TCP candidates in terrestrial plants and none in aquatic plants, and that TCP family genes originated early in the history of land plants. Phylogenetic analysis divided the candidate genes into Classes I and II, and Class II was further divided into CYCLOIDEA (CYC) and CINCINNATA (CIN) clades; CYC is more recent and originated from CIN in angiosperms. Protein architecture, intron pattern, and sequence characteristics were conserved in each class or clade supporting this classification. The two classes significantly expanded through whole-genome duplication during evolution. Expression analysis revealed the conserved expression of TCP genes from lower to higher plants. The expression patterns of Class I and CIN genes in different stages of the same tissue revealed their function in plant development and their opposite effects in the same biological process. Interaction network analysis showed that TCP proteins tend to form protein complexes, and their interaction networks were conserved during evolution. These results contribute to further functional studies on TCP family genes. Zea mays) [Antirrhinum majus) [Oryza sativa) [The plant-specific TCP transcription factor family genes are involved in multiple processes of plant growth and development, such as branching ,2, leaf ea mays) , CYCLOIDm majus) , and the sativa) ,11.Antirrhinum CINCINNATA (CIN) [AtBRC1 in Arabidopsis thaliana [OsFC1 in O. sativa [TgTB1 in Tulipa gesneriana [A. thaliana, AtTCP15 regulates internal replication and cytokinin and auxin responses during pistil development [AtTCP14 and AtTCP15 work together to regulate internode length [AtTCP23 participates in flowering time and plant development [The TCP proteins are categorized into two classes, Class I and Class II, according to the sequence homology of their TCP domains . Class ITA (CIN)  and the TA (CIN) . Class ITA (CIN) ,9,10,16.thaliana , OsFC1 isneriana  are invosneriana ,19. For elopment , AtTCP14e length  and seedelopment ,21. HowePhyscomitrella patens [Triticum aestivum) [A. thaliana [Populus euphratica [Gossypium raimondii [Given the critical roles of TCP genes in diverse biological processes, TCP homologous genes have been analyzed in various plants, and analyses of this gene family have been performed across species genomes. For instance, six TCP genes were identified in a patens , 29 in ma patens , 66 in westivum) , 24 in Aphratica , and 74 aimondii . These naimondii , none ofThus, to improve knowledge on the evolutionary history of the TCP gene family in plants, we performed a genome-wide systematic identification of the TCP proteins in the major plant lineages (47 species), including chlorophytes, bryophytes, gymnosperms, eudicots, and monocots. This allowed for a comprehensive assessment of their origin, evolution, classification, and patterns of differentiation and proliferation in various phylogenetic groups. Our gene structure, evolution, and expression analyses demonstrated that this gene family emerged at the very beginning of land plants’ establishment and expanded fast during higher plants’ evolution. The TCP genes in the same class or clade generally showed highly conserved gene/protein structure and expression profile across land plants, suggesting their common origin and functional conservation. Interaction network prediction showed that this gene family tends to form protein complexes involved in diverse biological processes, such as phytohormone pathways, cell cycles, and environmental responses.A. thaliana TCP proteins as queries. The 26 Viridiplantae included chlorophytes , the liverwort Marchantia polymorpha, mosses , the lycophyte Selaginella moellendorffii, Amborella trichopoda, eudicots , and monocots . We then performed BLASTP searches on another 21 aquatic plant genomes in the National Center for Biotechnology Information (NCBI) database, including Cyanophyta , Euglenophyta (Euglena gracilis and Colacium sideropus), Chrysophyta (Synura petersenii and Phaeothamnion confervicola), Chlorophyta , Charophyta , Phaeophyta (Ectocarpus siliculosus and Laminaria japonica), and Rhodophyta .To examine the distribution of TCP proteins in plants, we firstly implemented basic local alignment search tool searches (BLASTP)  on 26 ViM. polymorpha, but none were found in the 25 aquatic plants (B. napus showing the largest number of TCP proteins (75), whereas the lower plants M. polymorpha, S. fallax, P. patens, S. moellendorffii, and A. comosus had only two to nine TCP proteins . We identified 535 TCP proteins in 22 land plant species. These proteins were present in early-diverging land plants, such as c plants . One ricc plants . The genc plants . Overallc plants . No TCP c plants . In geneproteins .Taken together, our results showed that TCP proteins are present in terrestrial plants only and that these proteins rapidly expanded during the evolution from lower to higher plants, which is consistent with the increasing complexity of land plant genomes.To explore the evolutionary relationships between the 535 TCP proteins in land plants, we constructed a neighbor-joining (NJ) tree based on the alignment of the TCP domains using MEGA 5 .S. fallax, which had no Class II member. Class II was further divided into the CIN and CYC/TB1 clades based on tree topology and clade support values, with the CIN clade (179 members) being larger than the CYC clade (100 members). All of the land plants investigated had CIN clade TCP genes, while the CYC clade was only found in eudicots and monocots  and S. moellendorffii were clustered together into small branches of Classes I and II, respectively, as did the TCP proteins from monocots and eudicots; the TCP proteins of B. napus always clustered with those of their ancestral B. oleracea and B. rapa  . No specmonocots , suggest B. rapa .In summary, our phylogenetic analysis revealed that each gene class within the TCP family was conservatively distributed in land plants during their evolution and that Class I and CIN clade of Class II genes were old and emerged at the very beginning of land plants’ establishment.The DNA-binding domain (DBD) of TFs plays an important role in the recognition and binding of its target gene promoter sequences. To understand the sequence features in the DBD domains of TCP family genes, we performed a comprehensive analysis of the TCP domains across the 535 candidate TCP proteins, based on multiple alignments.N-terminal. However, the TCP domains of Class II CYC clade proteins were located at the middle of the candidates  residue at the 11th site in the RxRRxR region of Class I and the corresponding residue  at the 15th site of Class II accounted for the differences in binding targets between these two classes [AtTCP11 gene) obviously affected protein specificity [In general, the TCP domains of candidate proteins in Class I and Class II CIN clade were located at the ndidates . There wndidates . The TCPectively . In A. t classes . Moreovecificity . Accordicificity . The Lxxcificity . AccordiZ. marina , S. lycopersicum (SlTCP14 in Class I), S. tuberosum (StTCP1 in Class I), and Z. mays (ZmTCP41 in CYC clade). Except for ZmTCP41, the other five genes had one conserved intron insertion site and phases (phases 0) in the TCP domains  or after (in CIN clade and Class I) the TCP domains . MoreoveP domain . HoweverOverall, our results demonstrated that the protein structure of closely related members in a given clade were remarkably conserved across land plants, and that CIN and Class I TCP proteins were more similar in both motif distribution and structure, implying a common origin or close relationship.As mentioned above, the large number of TCP genes found in higher plants indicates this gene family underwent rapid expansion in land plant genomes during their evolution. To further examine the expansion mechanism of TCP genes, we used CoGe online software  to deterG. max, B. rapa, and B. napus syntenic duplication genes were ˃50%. Furthermore, most segmental duplication genes may be formed in ancestral genomes and be retained during whole-genome replication events. For example, in B. napus , most TCP genes (45.3%) derived from the recent hybridization event of the two ancestor diploid genomes  and Brassica oleracea ) ~75 million years ago, while only 6.6% genes originated from segmental duplication. Similar situations were observed in B. rapa and G. max. Polyploidy is a remarkable characteristic of higher plants’ chromosome evolution, and most extant angiosperms have experienced polyploidization at least once during their evolution [S. lycopersicum, G. max, and B. napus .MicroRNAs bind to specific genes as negative regulators, thus playing an important role in the regulation of the expressions of TF genes in plants . To explM. polymorpha, P. patens, and S. moellendorffii, the miR319 target genes were observed at the earliest in the basal angiosperm A. trichopoda , with only one G. max gene (GmTCP5) outside this subclade  had high expression levels in the archegoniophore, sporophyte, sporeling, and thallus, whereas MpTCP1 (Class II) had high expression levels in the antheridiophore, sporophyte, thallus, and gametophore   . The maj clades) .A. thaliana, it was reported that AtTCP2, AtTCP3, AtTCP4, AtTCP10, and AtTCP24 genes may indirectly regulate leaf development by regulating the boundary-specific genes CUC and LOB [G. max, O. sativa, Z. mays, and P. paten remain unknown  pathway  response, and salt, drought, and cold stresses [OsPCF2 and OsPCF6 participate in the regulation of salt stresses [A. thaliana, AtTCP14 and AtTCP15 were involved in defense responses against Hpa [Studies on Class I members are relatively fewer than those on Class II members. Class I members are also involved in regulating plant development, but have an opposite effect to those of Class II. Their best-known function is the regulation of leaf development: whereas Class I genes promote cell proliferation, CIN genes of Class II negatively regulate this process ,72. Moreaf shape ; the Chrgan size , the Phaelopment , and theelopment . Recentlelopment , and demstresses  while Osstresses ,78. In Ainst Hpa  and DC30inst Hpa . The divA. thaliana, AtTCP20 in Class I promotes auxin biosynthesis by promoting the expression of NRT.1 [AtTCP3 in CIN clade of Class II negatively modulates auxin response [AtTCP20 also inhibits the jasmonic acid (JA) biosynthesis [AtTCP4 in CIN clade affects JA biosynthesis in the opposite direction [It is interesting that both Class I and Class II genes were involved in hormone pathways, but they usually acted in opposite directions. For example, in of NRT.1 , while Aresponse ; AtTCP20ynthesis , whereasirection . Antagonirection  pathwaysA. thaliana, some TCP genes, such as AtTCP13 in the CIN clade and AtTCP14, 15, 19, and 21 in Class I, act as pathogen effector targets [O. sativa, OsTCP21 in the CIN clade participates in pathogen defense [S. lycopersicum, TCP14-2 (ortholog of AtTCP14) also contributed to enhance immunity to Phytophthora capsici [A. thaliana, SAP11 interacts with and destabilizes different members of CIN clade in class II, leading to the induction of severe leaf crinkling and down-regulation of jasmonic acid (JA) synthesis [Recent studies revealed that TCP genes play crucial roles in plant immunity. For example, in  targets . In O. s defense . In S. l capsici . Meanwhi capsici ,85,86. Fynthesis ,87. Receynthesis ,89. Notaynthesis ,89. To dynthesis , it is lIn summary, TCP genes are mainly involved in plant development, hormonal processes, and plant defense. The Class I and the CIN clade Class II genes had numerous functions whereas the CYC clade Class II genes had fewer roles. Moreover, the functions and expressions of CIN and CYC genes were obviously different, suggesting their functional divergence. Nevertheless, the Class I genes usually had opposite effect to Class II genes and this antagonism may be common in the plant kingdom during the process of plant evolution.In this study, we assessed the origin and evolutionary relationships of the TCP gene family across 47 plants ranging from algae to angiosperms using homology search of full-length proteins. Therefore, we could propose an evolutionary scenario for the TCP gene family .Klebsormidium nitens genome  were found in monocots and dicots, the CYC clade might have derived from the CIN clade before the divergence of dicots and monocots. Moreover, a recent study reported that PpTCP5 (CIN gene) is involved in regulating sporangium branching in P. patens [Most of the previous investigation in individual plant species ,111,112 . patens , similar. patens . A later. patens . Our phyB. napus, 52 genes (69%) derived from B. rapa (33 genes) and B. oleracea (19 genes) by a recent hybridization (about 75 million years ago) between these two ancestors. This is consistent with previous studies in which TF genes were preferentially retained after duplications during evolution [B. rapa  were maintained in B. napus while 50% genes from B. oleracea were lost, suggesting the tendency of gene retention after whole-genome duplication. Taken together, our results revealed that TCP genes mainly expanded through whole-genome duplication, and most of the duplicates were retained during land plants’ evolution.As plant complexity increased during the evolution, more intricate regulatory networks were needed. Gene duplication leads to the increase in the number of genes, thus providing the raw genetic resources for natural selection. Previous studies showed that segmental duplication contributed to the expansion of the TCP gene family . Howevervolution ,116. Morhttp://www.Phytozome.net) [A. thaliana TCP proteins [p < 0.1). After deleting the partial and redundant sequences, the TCP domains were identified in the candidates using ExPASy (http://expasy.org/prosite/) [B. oleracea (v 1.1) in BRAD [B. napus (v 1.0) in GENOSCOPE (http://www.genoscope.cns.fr/brassicanapus/) [https://blast.ncbi.nlm.nih.gov/Blast.cgi)  . For eacproteins  were userosite/) . The sam in BRAD  and B. nanapus/) . Moreoveast.cgi) . For balast.cgi) .−100 1E-100 and shared by about 70% members in a given class or clade were retained.Multiple alignments of the TCP domains of candidate proteins were performed in MAFFT 5 using the default parameters . Intron A NJ tree was constructed in MEGA 5  based onhttp://chibba.agtec.uga.edu/duplication/) [http://brassicadb.org/brad/) [https://genomevolution.org/coge/) [Syntenic blocks among the land plant species investigated in this study were downloaded from the PGDD (cation/) , BRAD (hg/brad/) , and CoGg/coge/)  databaseZ. mays (accession number: GSE27004) and O. sativa (accession number: GSE19024) were obtained from the Gene Expression Omnibus . The B. napus expression datasets were downloaded from the BioProject PRJNA358784 (NCBI database). The G. max expression dataset was obtained from Soybase (https://soybase.org/soyseq/). The P. patens expression dataset was acquired from BAR (http://bar.utoronto.ca/). The M. polymorpha expression dataset used was retrieved from a previous study [The expression datasets of us study . The heaus study . All genhttp://www.string-db.org) [http://services.cbu.uib.no/tools/kaks).The interaction network of TCP proteins was constructed based on the STRING 10.5 database (-db.org)  and disp-db.org) . To pred-db.org) . The pre


Alpinia oxyphylla Miq. (A. oxyphylla) is an important edible and traditional herbal medicine. In this study, the complete chloroplast genome of A. oxyphylla was sequenced, analysed, and compared to five species in the Zingiberaceae family. The size of the A. oxyphylla chloroplast genome was 161351 bp, which consisted of a large single-copy  and small single-copy  region separated by a pair of inverted repeats . The genome encoded 132 unique genes, including 87 protein-coding genes, 37 tRNAs and four rRNAs. The GC content of the genome was 36.17%. A total of 53 simple sequence repeats (SSRs) and 80 long repeats were identified in the A. oxyphylla chloroplast genome. The chloroplast genome of A. oxyphylla shared the highest sequence similarity of >90% with the chloroplast genome of A. zerumbet, and six chloroplast genomes in the Zingiberaceae family were compared by using CGView Comparison Tool (CCT). According to the phylogenetic tree, the Zingiberaceae family is divided into two categories, which coincide with the classification of the characteristics of sun-like and shade-like in plants. Our results reveal the phototrophic component of NADH-dehydrogenase (ndhB and ndhC), photosystem II (psbZ) and ATP synthase  exhibit adaptive evolution under different environments, and the strength of light is an important trigger for the adaptations at the chloroplast level. Alpinia oxyphylla Miq. (A. oxyphylla) is an important edible and traditional Chinese herbal medicine that originates in Hainan and is widely cultivated in southern China [A. oxyphylla have been used as valuable medicines that have a long clinical history and are often used as condiment food in China [A. oxyphylla is rich in flavonoids, sesquiterpenes, diterpenes, and diarylheptanoids, which have many pharmacological effects, such as improved memory, anti-oxidation, anti-inflammatory, neuroprotective and anticancer[rn China . The fruin China ,3. Numernticancer–8.Alpinia zerumbet, Amomum kravanh, Curcuma roscoeana, Curcuma flaviflora and Zingiber spectabile [A. oxyphylla chloroplast genome sequences have not yet been reported, which has seriously hindered the development of genetic diversity and breeding of Alpinia plants. Therefore, it is highly important and essential to study the phylogeny and evolution of Zingiberaceae plants [Chloroplasts are small organelles inside the cells of plants that contain photosynthetic machinery and produce essential energy for plants . Chloropectabile –15. A. oe plants .A. oxyphylla, including a description of its general features, IR contraction and expansion, codon usage and analysis of SSRs and long repeats. In addition, six chloroplast genome sequences in the Zingiberaceae family were compared by using CGView Comparison Tool (CCT). Moreover, we constructed a phylogenetic tree of the Zingiberales, which provides basic genetic information on the genetic diversity and breeding of Alpinia plants.In this study, we reported the complete chloroplast genome sequence of No specific permits were required for the collection of specimens for this study.A. oxyphylla leaves were collected from cultivated fields in Hainan Province, China. Total genomic DNA was extracted from 100 mg of fresh leaves using the Plant Genomic DNA Kit with a standard protocol . Purified genomic DNA was quantified with a Nanodrop 2000 spectrometer . Normalized genomic DNA was used to generate a 500 bp (insert size) paired-end library, following the Illumina HiSeq4000 standard protocol. Approximately 2 G raw data were generated with read lengths of 150 bp, and the chloroplast genome sequencing depth was nearly 60×.Fresh http://soap.genomics.org.cn/soapdenovo.html) on the basis of overlapping and paired-end relationships. Next, all clean reads were mapped onto the assembled contigs to obtain a complete chloroplast genome sequence. Genome confirmation was indispensable to perform after assembly. Finally, the paired-end clean reads were mapped onto the assembled genome with 100% coverage, and the insert-size matched the information of the sequenced library.First, Illumina paired-end reads were filtered on the basis of quality values, and the low-quality reads were trimmed. The remaining clean reads were used for assembly with SOAPdenovo2 (A. oxyphylla were further verified using the online tRNAscan-SE and tRNADB-CE search servers [A. oxyphylla chloroplast genome was drawn through Organellar Genome DRAW (OGDRAW v1.2) [Annotation was performed using the online program Dual Organellar GenoMe Annotator (DOGMA) . To prov servers –20. The AW v1.2) .A. oxyphylla chloroplast genome with five other chloroplast genomes. The boundaries between the IR and SC regions of A. oxyphylla and five other Zingiberaceae species were compared and analysed. The visualization of codon usage in the form of heatmaps of 17 species of Zingiberales and a histogram were conducted with R language with an RSCU value.The distribution of codon usage was detected by the software CodonW  with the relative synonymous codon usage (RSCU) ratio . The mVIA. oxyphylla. The length and identity of the repeats were limited to ≥ 30 bp and >90%, respectively [Repeat sequences in chloroplast genomes were detected by the REPuter program , includiectively . The SSRectively , with thectively .A. oxyphylla chloroplast genome was compared with other available chloroplast genomes of Zingiberales by using CCT [A. oxyphylla. The visualization of the circular map was conducted by CCT. AT distributions were measured on the basis of AT skewed using the equation: AT-skew = (A−T)/(A+T).The sing CCT . Genes wConcatenated alignments of 17 chloroplast genome sequences were performed using MUSCLE v.3.8.31. The phylogenetic analysis was carried out using the ML method with RAxML8.1, and the trees were visualized and annotated using the tree viewer of MEGA6 . StatistTo investigate the evolutionary process of light adaptation of Zingiberaceae plants, we calculated the Nonsynonymous (Ka), Synonymous (Ks) and Ka/Ks ratios of protein coding genes associated with the photosystem using KaKs_Calculator 2.0 .A. oxyphylla (GenBank Accession Number: KY985237) has a typical quadripartite structure and is a circular molecule 161,351 bp in size  is the longest, and the Z. spectabile chloroplast genome (155890 bp) is the shortest. Interestingly, the SSC region  of A. kravanh is the shortest, whereas the SSC region (18611 bp) of the Z. spectabile chloroplast genome is the longest. Five of the chloroplast genomes contain 132 genes, excluding only A. kravanh (135 genes). As shown in C. roscoeana chloroplast genome has the highest GC content (36.33%), while the A. kravanh chloroplast genome has the lowest GC content (31.3%). In addition, 87 protein genes were identified in A. oxyphylla, 80 were identified in A. kravanh, and 86 were identified in the other four species. Four conserved rRNAs were identified in every species. The A. oxyphylla chloroplast genome encodes 37 types of tRNAs, A. zerumbet, C. roscoeana, C. flaviflora and Z. spectabile encode 38, whereas A. kravanh encodes 30 , IR region (15.09%) and SSC region (9.43%), respectively  were compared, and the results suggested that there was little difference in the distribution pattern and number of SSRs among the six chloroplast genomes  repeats, 36 palindrome (inverted) repeats and 14 reverse repeats (A. oxyphylla and its related species [Simple sequence repeats (SSRs), also known as microsatellites, are a group of tandem repeated sequences, generally ranging in length from 1–6 or more base pairs and are widely distributed in chloroplast genomes . A totalectively . Further genomes . In addi repeats . Fifty-s species ,39.A. oxyphylla chloroplast genome was compared with 16 previously reported chloroplast genomes of Zingiberales by using CCT  bootstrap values had values of 100% bootstrap support for the Zingiberaceae family except the node of Z. spectabile and C. flaviflora (92%)  . The A. A. oxyphylla, A. kravanh and A. zerumbet clustered into one branch, C. roscoeana, C. flaviflora and Z. spectabile clustered into another , photosystem II (psbZ) and ATP synthase  exhibit adaptive evolution under different environments.The complete chloroplast sequence of S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Clostridioides difficile occurs in up to half of infants under the age of 3 months, is strongly influenced by feeding modality and is largely asymptomatic. In spite of this, C. difficile's presence has been associated with susceptibility to chronic disease later in childhood, perhaps by promoting or benefiting from changes in infant gut microbiome development, including colonization with pathogenic bacteria and disrupted production of microbial bioactive metabolites and proteins. In this study, the microbiomes of 1554 infants from the CHILD Cohort Study were described according to C. difficile colonization status and feeding mode at 3–4 months of age. C. difficile colonization was associated with a different gut microbiome profile in exclusively breastfed (EBF) vs. exclusively formula fed (EFF) infants. EBF infants colonized with C. difficile had an increased relative abundance of Firmicutes and Proteobacteria, decreased relative abundance of Bifidobacteriaceae, greater microbiota alpha-diversity, greater detectable fecal short chain fatty acids (SCFA), and lower detectable fecal secretory Immunoglobulin A (sIgA) than those not colonized. Similar but less pronounced differences were seen among partially breastfed infants (PBF) but EFF infants did not possess these differences in the gut microbiome according to colonization status. Thus, breastfed infants colonized with C. difficile appear to possess a gut microbiome that differs from non-colonized infants and resembles that of EFF infants, but the driving force and direction of this association remains unknown. Understanding these compositional differences as drivers of C. difficile colonization may be important to ensure future childhood health.Colonization with  Clostridioides (formerly Clostridium) difficile is a bacterium that is present in the intestine of nearly 40% of infants at 1 month of age, and 30% of infants between the ages of 1 and 6 month   , one stu feeding , 9, 10. man milk –13. ConsC. difficile (family Peptostreptococcaceae) and other gut microbiome components, including composition, metabolites and sIgA, to provide insights into ecological factors related to C. difficile expansion in infancy. We also explored these differences in exclusively breastfed, partially breastfed, and exclusively formula fed infants to examine the gut microbial community and C. difficile colonization infants with distinct diets.In this study, we report the association between www.childstudy.ca)  and secretory IgA (n = 731)  . The pritudy.ca) . All infn = 731) . Gut micn = 731) , but thiC. difficile and followed the methods set by Penders et al. . Amongst colonized infants, median levels of C. difficile (ng/g feces) in infant fecal samples were not different between feeding groups (data not shown).Fecal samples of 5–10 grams were collected from infant diapers during home-visits conducted at 3–4 months of age by a research assistant or parents according to an approved protocol . Sampless et al. . To minis et al. . Then, eDNA extraction and amplification of bacterial V4 hypervariable region of the bacterial 16S rRNA gene was followed by sequencing and taxonomic classification and was conducted as previously described . To summN = 467), metabolites were quantified by magnetic resonance spectroscopy (NMR). NMR requires a small quantity of sample for processing and has high reproducibility compared to mass spectrometry (2O: 46% 750 mM potassium phosphate (mono- and dibasic) pH 7.0 v/v containing 5 mM DSS-d6 . The sample (350 μL) was then transferred to 3 mm SampleJet NMR tube for subsequent spectral analysis. All 1H-NMR spectra were collected on a 700 MHz Avance III (Bruker) spectrometer equipped with a 5 mm HCN Z-gradient pulsed-field gradient (PFG) cryoprobe. 1H-NMR spectra were acquired at 25°C using the first transient of the NOESY pre-saturation pulse sequence (noesy1dpr), chosen for its high degree of quantitative accuracy.In a sub-set of fecal samples   were zero-filled to 250 K data points and line broadened 0.5 Hz. The methyl singlet produced by a known quantity of DSS was used as an internal standard for chemical shift referencing (set to 0 ppm) and for quantification. All ton, AB) . The Chelutamate .N = 731) using the Secretory IgA ELISA (enzyme-linked immunosorbent assay) kit . Approximately 14 mg of fecal sample was used for the sIgA analyses. Samples were run in duplicate according to the manufacturer's protocol, as previously described . A 3-category variable was created for infant breastfeeding status at the time of stool sample collection and questionnaire administration: (1) exclusively breastfed (EBF), (2) partially  breastfed (PBF), and (3) exclusively formula fed (EFF). Complete feeding data were missing in 8 infants, leaving a total of 1554 infants  that were stratified by feeding mode.Breastfeeding status was determined through self-report questionnaires administered to mothers at 3–4 months postpartum (t-test) tests were used where appropriate (C. difficile colonization status (predictor) were determined using the multivariate association with linear models method developed by the Huttenhower lab (MaAslin) . Spearman correlations were computed in p or q-value < 0.05, after FDR correction for multiple comparisons.All statistical analysis was conducted using Stata (version 13), RStudio (version 1.1.456), and the Galaxy platform (MaAslin) between September 2018 and March 2019. Non-parametric  and parametric (student's ropriate  to compaMaAslin)  , which aligns with previously reported estimates . The mean Shannon and Chao1 indices for EBF and PBF infants were lower for infants who lacked C. difficile compared to infants colonized with C. difficile, suggesting that the richness and abundance of the infant gut microbiota are greater and more equally distributed in the presence of C. difficile ] in each of the feeding groups was similar: 3.29 months (1.03) for EBF, 3.33 months (0.94) for PBF, and 3.20 months (1.10) for EFF, p = 0.27.The prevalence of  n = 482/562, whicBifidobacterium spp. , Peptostreptococcaceae  and Clostridiaceae  families . Propionate production by Lachnospiraceae is through the 1,2-propanediol and acrylate pathways, which are possessed by Blautia, Eubacterium, and Coprococcus , Lachnospiraceae  and Clostridiaceae  were all positively correlated with butyrate and enriched in PBF infants . Since Bacteroidaceae are more abundant with any formula feeding  infants and equally no differences were detected in fecal metabolites. The sole family of microbes whose relative abundance was significantly higher in EFF C. difficile carriers was its own family, the Peptostreptococcaceae   , 33. Indla spp.) .C. difficile was associated with lower sIgA concentrations among EBF infants   infants colonized with um spp.) . Meta-anum spp.) . Extendi infants .C. difficile. Should our study findings continue to align with previous findings, we might expect a prevalence of toxigenic strains to be <10% among infants with C. difficile positive samples  to assess if C. difficile colonization is transient or persistent and whether the microbiome changes precede or follow colonization. However, with enhanced characterization of the gut microbiome beyond taxon composition, our study provides evidence for a putative role of C. difficile colonization on the gut microbial ecology of young, full-term infants from a large, general population in North America.In our large population cohort study, we were not able to categorize infants according to the proportional intake of breastmilk vs. formula, as others have . Since o samples , 41. We C. difficile and this distinction depended on the breastfeeding status of the infant. The most noticeable microbiome differences with C. difficile colonization, especially depletion of Bifidobacterium spp., were among EBF infants. Similar compositional differences among members of the Firmicutes phylum were seen in EBF and PBF infants. However, unique to PBF infants was enrichment of Veillonellaceae. These findings highlight the differential relationship of C. difficile colonization on EBF vs. PBF vs. EFF infants, which should be considered in future studies of infants feeding modality and disease risk. In summary, we found differences in the infant gut microbiome with C. difficile colonization, but it remains unclear whether C. difficile causes these differences or if external factors in early infancy create a niche that is more permissive to colonization. Newer cohorts with available multi-omics data could validate these findings and explore the hypothesized relations between various microbiota and C. difficile to further understand colonization of this microbe in infancy and its implications in later childhood health.We observed a distinct gut microbiome in young infants colonized with CHILDdb.ca database.The data and analysis code that support the findings of this study can be made available from the corresponding author and CHILD Cohort Study coordinators upon reasonable request. These data, including study participant data, are securely stored in the The studies involving human participants were reviewed and approved by the University of Alberta, University of British Columbia and University of Manitoba Ethics Boards. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.C. difficile detection. DW and RM supervised and conducted NMR and fecal metabolite analyses. CF supervised, conducted, and helped interpret the fecal sIgA analyses. MA created the breastfeeding measures. DG, AB, PM, PS, ST, TM, MS, DL, and JS obtained funding and contributed to study design and data collection. AK obtained funding, contributed to data interpretation and critically reviewed the manuscript. All the authors reviewed the manuscript content, provided feedback and approved the final version. AK will serve as guarantor of the manuscript's contents.KD and AK conceived the study. KD performed data analysis, prepared figures, and drafted and edited the manuscript. HT generated gut microbiota operational taxonomic unit profiles using QIIME software. TK conducted DNA extraction and sample preparation for sequencing for microbiome analyses. NM-L performed targeted qPCR for The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.