BMC Microbiology

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The effects of cropping practices on the rhizosphere soil physical properties and microbial communities of Bupleurum chinense have not been studied in detail. The chemical properties and the microbiome of rhizosphere soil of B. chinense were assessed in the field trial with three cropping practices (continuous monocropping, Bupleurum-corn intercropping and Bupleurum-corn rotation). The results showed cropping practices changed the chemical properties of the rhizosphere soil and composition, structure and diversity of the rhizosphere microbial communities. Continuous monocropping of B. chinense not only decreased soil pH and the contents of NO3⁻-N and available K, but also decreased the alpha diversity of bacteria and beneficial microorganisms. However, Bupleurum-corn rotation improved soil chemical properties and reduced the abundance of harmful microorganisms. Soil chemical properties, especially the contents of NH4⁺-N, soil organic matter (SOM) and available K, were the key factors affecting the structure and composition of microbial communities in the rhizosphere soil. These findings could provide a new basis for overcoming problems associated with continuous cropping and promote development of B. chinense planting industry by improving soil microbial communities.
 
Background Pseudomonas spp. promotes plant growth and colonizes a wide range of environments. During the annotation of a Coffea arabica ESTs database, we detected a considerable number of contaminant Pseudomonas sequences, specially associated with leaves. The genome of a Pseudomonas isolated from coffee leaves was sequenced to investigate in silico information that could offer insights about bacterial adaptation to coffee phyllosphere. In parallel, several experiments were performed to confirm certain physiological characteristics that could be associated with phyllospheric behavior. Finally, in vivo and in vitro experiments were carried out to verify whether this isolate could serve as a biocontrol agent against coffee rust and how the isolate could act against the infection. Results The isolate showed several genes that are associated with resistance to environmental stresses, such as genes encoding heat/cold shock proteins, antioxidant enzymes, carbon starvation proteins, proteins that control osmotic balance and biofilm formation. There was an increase of exopolysaccharides synthesis in response to osmotic stress, which may protect cells from dessication on phyllosphere. Metabolic pathways for degradation and incorporation into citrate cycle of phenolic compounds present in coffee were found, and experimentally confirmed. In addition, MN1F was found to be highly tolerant to caffeine. The experiments of biocontrol against coffee leaf rust showed that the isolate can control the progress of the disease, most likely through competition for resources. Conclusion Genomic analysis and experimental data suggest that there are adaptations of this Pseudomonas to live in association with coffee leaves and to act as a biocontrol agent.
 
Background The orchid growth and development often associate with microbes. However, the interaction between plant performance and microbial communities within and surrounding plants is less understood. Dendrobium catenatum , which used to be an endangered orchid species, has become a billion dollar industry in China. Simulated natural cultivation modes, such as living tree epiphytic (LT) and cliff epiphytic (CE) cultivations, improve the production or quality of D. catenatum and contribute to the development of D. catenatum industry. In a previous study, morphological characteristics, anatomical structure, and main bioactive components (polysaccharides and ethanol-soluble extractives) of D. catenatum grown under LT and CE significantly differed from a facility cultivation mode, pot (PO) cultivation, were observed. Whether cultivation mode affects bacterial and fungal communities of D. catenatum , thereby affecting the chemical quality of this plant, need to be explored. Results Both three plant organs (leaf, stem, and root) and cultivating substrates obtained under three cultivation modes: living tree epiphytic (LT), cliff epiphytic (CE), and pot (PO) cultivation were examined by adopting high-throughput sequencing methods. Subsequently, bacterial and fungal correlations with D. catenatum main chemical components, stem polysaccharides and ethanol-soluble extractives and leaf phenols and flavonoids, were elucidated. The results showed that microbial communities of the plants and substrates are both influenced by the cultivation mode. However, the plants and their cultivating substrates exhibited different patterns of bacterial and fungal composition, with clearly distinguished dominant bacterial groups, but shared dominance among fungal groups. Bacteria and fungi differed in abundance, diversity, and community structure, depending on the cultivation environment and plant organ. Both bacterial and fungal communities were affected by cultivation mode and plant organ. In both plants and substrates, PO bacterial and fungal community structure differed significantly from those of LT and CE modes. Bacterial and fungal community structure differed significantly between roots and the other two plant organs examined (stems and leaves). Several bacteria and fungi were positively correlated with main chemical components in D. catenatum . Conclusions The findings indicate that microbial communities of the plants and substrates were both influenced by the cultivation mode and plant organ, and some of them were positively correlated with main chemical components in D. catenatum . The research would enhance our understanding of interactions between Dendrobium and the microbial environment, and to provide a theoretical basis for the development of improved D. catenatum cultivation methods.
 
Background The widespread use of antibiotics has led to the emergence of many drug-resistant strains; thus, the development of new antibacterial drugs is essential with antimicrobial peptides becoming the focus of research. This study assessed the antibacterial effect of a novel antimicrobial peptide, named LL-1 on Escherichia coli (E.coli) by determining the minimum inhibitory concentration (MIC) and the antibacterial curve. The interaction between LL-1 and E. coli DNA was then detected by nucleic acid gel electrophoresis. The effect of LL-1 on the E. coli cell membrane was assessed by detecting the leakage of β-galactosidase, nucleic acid and protein. The influence of LL-1 on the intracellular ATP of E. coli was analysed by determining the concentration of intracellular ATP. Finally, the bacteria and colonies of E. coli treated with LL-1 were observed using scanning and transmission electron microscopy. Results The results suggested that the MIC value was 3.125 µg/ml, and the antibacterial effect was dose-dependent. LL-1 dose-dependently combined with E. coli DNA. LL-1 resulted in the leakage of intracellular β-galactosidase, nucleic acid and protein, and decreased intracellular ATP concentrations of E. coli. Two MIC of LL-1 caused E. coli to shrink, resulting in a rough surface, plasmolysis, and bacterial adhesion. Conclusion This study indicated that LL-1 had a good bactericidal effect on E. coli by mainly increasing the permeability of the cell membrane, leading to leakage of the intracellular content. This will lay the foundation for an in-depth study on the antibacterial mechanism of LL-1 against E. coli and its clinical application.
 
Anti-Panton-Valentine-leukocidin (PVL)-antibodies in Africans compared to Germans. Serum levels of anti-PVL-antibodies (a) and anti-Hla-antibodies (b) of participants from Germany and Africa were quantified by ELISA and expressed in arbitrary units (AU). Median values for levels of antibodies to PVL in Caucasians and Africans were 0.7 AU (interquartile range (IQR), 0.5–1.1 AU) and 1.9 AU (IQR, 1.4–2 AU), respectively. Median values for levels of antibodies to Hla in Caucasians and Africans were 29.1 AU (IQR, 20.1–40.2 AU) and 42.4 AU (IQR, 30.8–50 AU), respectively. c The titers of antibodies against PVL were corrected for their respective anti-Hla titers and displayed as fold-change to median serum level of Caucasians (IQR, 0.7–1.4). Median value for relative anti-PVL titers in Africans was 1.6 (IQR, 1.2–2). Statistical significance was analyzed by Mann-Whitney test or unpaired t test (** p < 0.01, *** p < 0.001, **** p < 0.0001)
Isolated polymorphonuclear leukocytes (PMNs) from the German and African donor were subjected to 5 nM recombinant Panton-Valentine leukocidin (PVL) after pre-incubation with serial dilutions of pooled serum from German or African participants. Neutralization of the cytotoxic effect of PVL is displayed as percentage of undamaged PMNs. Half-maximal (50%) and 90% inhibition thresholds are indicated as dotted lines
Background The prevalence of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin (PVL) gene is higher in Africa (≈50%) compared to Europe (< 5%). The study aimed to measure anti-PVL-antibodies in Africans and Germans in a multi-center study and to test whether detected antibodies can neutralize the cytotoxic effect of PVL on polymorphonuclear leukocytes (PMNs). Methods Sera from asymptomatic Africans ( n = 22, Nigeria, Gabon) and Caucasians ( n = 22, Germany) were used to quantify antibody titers against PVL and α-hemolysin (in arbitrary units [AU]) by ELISA. PMNs from one African and German donor were exposed to 5 nM recombinant PVL to measure the neutralizing effect of serial dilutions of pooled sera from African and Caucasian participants, or donor sera at 0.625 and 2.5% (v/v). Results Anti-PVL-antibodies were significantly higher in Africans than in Germans (1.9 vs. 0.7 AU, p < 0.0001). The pooled sera from the study participants neutralized the cytotoxic effect of PVL on African and German PMNs in a dose dependent manner. Also, neutralization of PVL on PMNs from the African and German donors had a stronger effect with African sera (half-maximal inhibitory concentration (IC 50 ) = 0.27 and 0.47%, respectively) compared to Caucasian sera (IC 50 = 3.51 and 3.59% respectively). Conclusion Africans have higher levels of neutralizing anti-PVL-antibodies. It remains unclear if or at what level these antibodies protect against PVL-related diseases.
 
Background Rhizosphere microorganisms play a crucial role in plant health and development. Plant root exudates (PRE) are a complex mixture of organic molecules and provide nutritional and signaling information to rhizosphere microorganisms. Burkholderiaceae species are non-abundant in the rhizosphere but exhibit a wide range of plant-growth-promoting and plant-health-protection effects. Most of these plant-associated microorganisms have been studied in isolation under laboratory conditions, whereas in nature, they interact in competition or cooperation with each other. To improve our understanding of the factors driving growth dynamics of low-abundant bacterial species in the rhizosphere, we hypothesized that the growth and survival of four Burkholderiaceae strains ( Paraburkholderia phytofirmans PsJN, Cupriavidus metallidurans CH34, C. pinatubonensis JMP134 and C. taiwanensis LMG19424) in Arabidopsis thaliana PRE is affected by the presence of each other. Results Differential growth abilities of each strain were found depending on plant age and whether PRE was obtained after growth on N limitation conditions. The best-adapted strain to grow in PRE was P. phytofirmans PsJN, with C. pinatubonensis JMP134 growing better than the other two Cupriavidus strains. Individual strain behavior changed when they succeeded in combinations. Clustering analysis showed that the 4-member co-culture grouped with one of the best-adapted strains, either P. phytofirmans PsJN or C. pinatubonensis JMP134, depending on the PRE used. Sequential transference experiments showed that the behavior of the 4-member co-culture relies on the type of PRE provided for growth. Conclusions The results suggest that individual strain behavior changed when they grew in combinations of two, three, or four members, and those changes are determined first by the inherent characteristics of each strain and secondly by the environment.
 
Probiotics contain beneficial live bacteria that confer several health benefits to the host. For the past 50 years, spore-forming Bacillus species have been used in the form of probiotics. Among these, Bacillus clausii strains are used for the management of acute and antibiotic-associated diarrhoea. In the present work, we have evaluated the asserted label information on randomly chosen commercial Bacillus clausii spore suspension of probiotic products. The quality and number of viable bacteria were evaluated based on the colony count, antibiotic resistance, and hemolytic activity assays. The colony fingerprinting and 16S rRNA gene-sequencing techniques were used to confirm the presence of a univariate strain (Bacillus clausii). Our results corroborated the label count of 2 × 10 9
 
Objectives Colibacillosis is a frequent enteric disease in the pig industry that causes significant economic losses. The objective of this study was to investigate the molecular characteristics of fluoroquinolone (FQ)-resistant E. coli isolates from suckling piglets with colibacillosis. Results A total of 43 FQ-resistant E. coli isolates were tested in this study and all isolates showed multi-drug resistance (MDR) and mutations in quinolone resistance determining regions (gyrA or parC). Especially, FQ-resistant E. coli isolates with double mutations in both gyrA and parC were shown a high FQs minimum inhibitory concentration (≥ 64 mg/L for ciprofloxacin, ≥ 128 mg/L for enrofloxacin, and ≥ 256 mg/L for norfloxacin). Among 43 FQ-resistant E. coli isolates, 12 (27.9%) were showed plasmid-mediated quinolone resistance (PMQR) positive E. coli. Prevalence of PMQR gene, aac(6’)-Ib-cr, qnrS, and qepA, were identified in 7, 3, and 2 E. coli isolates, respectively. We identified the following in PMQR-positive E. coli isolates: the tetracycline resistance genes tetD (12 isolates, 100.0%), tetE (12 isolates, 100.0%), tetA (11 isolates, 91.7%), and tetB (1 isolate, 8.3%); β-lactamases–encoding blaCMY-2 (10 isolates, 83.3%), blaTEM-1 (7 isolates, 58.3%), blaOXA-1 (7 isolates, 58.3%), blaSHV-1 (3 isolates, 16.7%), and blaAAC-2 (1 isolate, 8.3%); and the chloramphenicol resistance genes (10 isolates, 83.3%); the sulfonamide resistance genes sul1 (9 isolates, 75.0%) and sul2 (10 isolates, 83.3%); the aminoglycoside modifying enzyme gene aac(3)-II (2 isolates, 16.7%). The F4 (7 isolates, 58.3%), LT:STb:EAST1 (5 isolates, 41.7%), and paa (3 isolates, 25.0%) were most common fimbrial antigen, combinations of toxin genes, and non-fimbrial adhesins genes, respectively. All PMQR-positive E. coli carried class I integrons but only 4 isolates carried the gene cassette. The most prevalent plasmid replicon was FIB (9 isolates, 75.0%), followed by FIC, HI1, and N (7 isolates, 58.3%), respectively. Conclusions Because FQ-resistant E. coli can serve as a reservoir of FQ resistant genetic determinants that can be transferred to pathogenic bacteria in humans or pigs, this represents a public health hazard.
 
Background Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen and has emerged as one of the most troublesome pathogens. Drug resistance in A. baumannii has been reported on a global scale. Minocycline was found to be active against multi-drug resistant A. baumannii and was approved by the FDA for the infections caused by sensitive strains of A. baumannii. However, the emergence of minocycline resistance and its toxic effects still need to be addressed. Therefore, this study aimed to evaluate the synergistic effects of metformin combined with minocycline on minocycline-resistant A. baumannii . Results The effect of metformin on the antibacterial activity of minocycline was determined by checkerboard and time-killing assay. Further, it was observed by biofilm formation assay that metformin combination with minocycline can inhibit the formation of biofilm. Outer membrane integrity, membrane permeability, membrane potential and reactive oxygen species (ROS) were monitored to explore the underlying synergistic mechanisms of metformin on minocycline. And the results shown that metformin can destroy the outer membrane of A. baumannii , enhance its membrane potential, but does not affect the membrane permeability and ROS. Conclusion These findings suggested that the combination of metformin and minocycline has the potential for rejuvenating the activity of minocycline against minocycline-resistant A. baumannii .
 
Chinese medicine tongue coating types and photos. Tongue coating: a layer of moss-like material covering the tongue. TWF:thin white tongue fur; TYF:thin yellow tongue fur; WGF:white greasy tongue fur; YGF:yellow greasy tongue fur
Analysis of microbial a-diversity of four different types of tongue coating. A Rarefaction curve: the abscissa is the number of sequencing strips randomly selected from a sample, and the ordinate is the number of OTUs that can be constructed based on the number of sequencing strips, which is used to reflect the sequencing depth. Different samples are represented by curves of different colors. B Venn diagram of OTUs with four different tongue coatings.The Veen diagram depicts the number of OTUs specific and shared by each of the four tongue coatings TWF, TYF, YGF, and WGF. C and D Results of a-diversity analysis of four tongue coatings. C Simpson index; D Shannon index
Analysis of microbial β-diversity of four different types of tongue coatings A and B Results of β-diversity analysis of four tongue coatings. A unweighted Unifrac distances and (B) weighted Unifrac distances. C and D Cumulative graph of microorganisms with percentages greater than 1 in four different tongue coatings. C Genus level and (D) Species level. E and F The differences in microbial abundance between TWF and the remaining three tongue coating types at the species level showed statistically significant results. E TWF-TYF, (F) TWF-YGF and (G) TWF-WGF
Discrepancies of co-occurrence networks of the four different tongue coating types A, B, C and D The complex nature of inter microbial interactions in the ecological community of each tongue coating was characterized by co-occurrence networks using graphs. Main groups of co-occurrence species are presented in different colours, and smaller groups are shown in grey. Networks of species in the four tongue coatings are identified by positive and negative correlations among the dominant bacteria. Red line indicates positive correlation and blue line indicates negative correlation. Only the bacterial connections (edges) larger than cut-offs (correlation values > 0.4) are retained. Each node in the network indicates a species. The size of each node is proportional to the relative abundance of each species. Nodes in red color show driver microbes which significantly contributed to the separation of the networks (NESH-score value of > 2). E, F and G Network view: All nodes belonging to the same community are randomly assigned similar colors, and gray nodes represent nodes existing in both case and control subnets. The node size is directly proportional to its nesh score. If the nesh score of a node is case > control, it will be marked in red. Therefore, a large red node can be regarded as a driving node. For edges, red indicates the interaction that exists only in the case subnet, green indicates the interaction that exists only in the control subnet, and blue indicates the interaction that exists in both subnets
A The effect of clinical indicators and demographic variables on the microbial a-diversity of the tongue, with red representing positive correlations and blue representing negative correlations.” + ” indicates p < 0.05,” +  + ” indicates p < 0.001. B Contributions of clinical and demographic variables to the differences in relative abundances of key distinguishing bacterial species for four tongue coatings based on Spearman correlation coefficients and best multiple regression model. Color bar shows correlation values, where red color indicates positive association, blue color negative association, and only significant correlations were shown.” + ” indicates p < 0.05,” +  + ” indicates p < 0.001
Background: Tongue coating is an important health indicator in traditional Chinese medicine (TCM). The tongue coating microbiome can distinguish disease patients from healthy controls. To study the relationship between different types of tongue coatings and health, we analyzed the species composition of different types of tongue coatings and the co-occurrence relationships between microorganisms in Chinese adults. From June 2019 to October 2020, 158 adults from Hangzhou and Shaoxing City, Zhejiang Province, were enrolled. We classified the TCM tongue coatings into four different types: thin white tongue fur (TWF), thin yellow tongue fur (TYF), white greasy tongue fur (WGF), and yellow greasy tongue fur (YGF). Tongue coating specimens were collected and used for 16S rRNA gene sequencing using the Illumina MiSeq system. Wilcoxon rank-sum and permutational multivariate analysis of variance tests were used to analyze the data. The microbial networks in the four types of tongue coatings were inferred independently using sparse inverse covariance estimation for ecological association inference. Results: The microbial composition was similar among the different tongue coatings; however, the abundance of microorganisms differed. TWF had a higher abundance of Fusobacterium periodonticum and Neisseria mucosa, the highest α-diversity, and a highly connected community (average degree = 3.59, average closeness centrality = 0.33). TYF had the lowest α-diversity, but the most species in the co-occurrence network diagram (number of nodes = 88). The platelet-to-lymphocyte ratio (PLR) was associated with tongue coating (P = 0.035), and the YGF and TYF groups had higher PLR values. In the co-occurrence network, Aggregatibacter segnis was the "driver species" of the TWF and TYF groups and correlated with C-reactive protein (P < 0.05). Streptococcus anginosus was the "driver species" in the YGF and TWF groups and was positively correlated with body mass index and weight (P < 0.05). Conclusion: Different tongue coatings have similar microbial compositions but different abundances of certain bacteria. The co-occurrence of microorganisms in the different tongue coatings also varies. The significance of different tongue coatings in TCM theory is consistent with the characteristics and roles of the corresponding tongue-coating microbes. This further supports considering tongue coating as a risk factor for disease.
 
Background: Rumen microbes play an important role in ruminant energy supply and animal performance. Previous studies showed that the rumen microbiome of Mongolian cattle has adapted to degrade the rough forage to provide sufficient energy to tolerate the harsh desert ecological conditions. However, little is known about the succession of rumen microbes in different developmental stages of post-weaning Mongolian cattle. Methods: Here, we examined the succession of the rumen microbial composition and structure of 15 post-weaning Mongolian cattle at three developmental stages i.e., 5 months (RM05), 18 months (RM18) and, 36 months (RM36) by using the 16S rRNA gene sequencing method. Results: We did not find any age-dependent variations in the ruminal concentrations of any volatile fatty acid (VFA) of Mongolian cattle. The diversity of the rumen bacterial community was significantly lower in RM05 group, which reached to stability with age. Bacteroidetes and Firmicutes were the two dominant phyla among all age groups. Phy-lum Actinobacteria was significantly higher in RM05 group, phyla Spirochaetes, and Tenericutes were highly abundant in RM18 group, and phyla Proteobacteria and Epsilonbacteraeota were enriched in RM36 group. Genera Prevotella_1, Bacteroides, and Bifidobacterium were abundant in RM05 group. The short chain fatty acid (SCFA) producing bacteria Rikenellaceae_RC9_gut_group showed high abundance in RM18 group and fiber degrading genus Alloprevotella was highly abundant in RM36 group. Random forest analysis identified Alloprevotella, Ileibacterium, and Helicobacter as important age discriminatory genera. In particular, the genera Ruminococcaceae_UCG-005, Bacteroides, Saccha-rofermentans, and Fibrobacter in RM05, genera [Eubacterium] coprostanoligenes_group, Erysipelotrichaceae_UCG-004, Helicobacter, Saccharofermentans, Papillibacter, and Turicibacter in RM18, and genera Rikenellaceae_RC9_gut_group, Lachnospiraceae_AC2044_group, and Papillibacter in RM36 showed the top interactions values in the intra-group interaction network. Conclusions: The results showed that rumen microbiota of Mongolian cattle reached to stability and maturity with age after weaning. This study provides some theoretical evidence about the importance of functional specific rumen bacteria in different age groups. Further studies are needed to determine their actual roles and interactions with the host.
 
Background Bone loss and deformation due to damage caused by injury or recurrent invasive infections presents a major clinical obstacle. While bone substitute biomaterials promote osseous tissue regeneration, their application in sites complicated by microbial infections such as osteomyelitis, is limited. Bioactive glass biomaterials (Bioglass) have been shown to have efficient mechanisms of repairing the integrity of bone, while inhibiting growth of a range of bacterial strains. There are several commercially available bioactive glass compounds, each with a unique chemical composition. One compound in particular, S53P4, has demonstrated antimicrobial effects in previous studies but the antimicrobial activity of the parent compound 45S5 has not been investigated. Results To assess whether antimicrobial activity is common among bioglass compounds, 45S5-the parent compound, was evaluated in comparison to S53P4 for antibacterial and antibiofilm effects against multiple strains of aerobic and anaerobic bacteria associated with various types of osteomyelitis. Experiments of antimicrobial effects in liquid cultures demonstrated that both compounds were antimicrobial against various microbial genera including S. gordonii , V. parvula , P. aeruginosa and MRSA; particles of the smallest size (32–125 µm) invariably showed the most robust antimicrobial capabilities. When employed against biofilms ecological biofilms grown on hydroxyapatite, 45S5 particles produced a stronger reduction in biofilm mass compared to S53P4 particles when considering small particle ranges. Conclusion We found that 45S5 seems to be as effective as S53P4 and possibly even more capable of limiting bacterial infections. The efficacy of bioactive glass was not limited to inhibition of planktonic growth, as it also extended to bacterial biofilms. The increased antibacterial activity of 45S5 compared to S53P4 is true for a variety of size ranges.
 
Macrophages play an essential role in the process of recognition and containment of microbial infections. These immune cells are recruited to infectious sites to reach and phagocytose pathogens. Specifically, in this article, bacteria from the genus Mycobacterium , Salmonella and Escherichia , were selected to study the directional macrophage movement towards different bacterial fractions. We recreated a three-dimensional environment in a microfluidic device, using a collagen-based hydrogel that simulates the mechanical microarchitecture associated to the Extra Cellular Matrix (ECM). First, we showed that macrophage migration is affected by the collagen concentration of their environment, migrating greater distances at higher velocities with decreasing collagen concentrations. To recreate the infectious microenvironment, macrophages were exposed to lateral gradients of bacterial fractions obtained from the intracellular pathogens M. tuberculosis and S. typhimurium . Our results showed that macrophages migrated directionally, and in a concentration-dependent manner, towards the sites where bacterial fractions are located, suggesting the presence of attractants molecules in all the samples. We confirmed that purified M. tuberculosis antigens, as ESAT-6 and CFP-10, stimulated macrophage recruitment in our device. Finally, we also observed that macrophages migrate towards fractions from non-pathogenic bacteria, such as M. smegmatis and Escherichia coli . In conclusion, our microfluidic device is a useful tool which opens new perspectives to study the recognition of specific antigens by innate immune cells.
 
Background To explore and utilize abundant soil microbes and their beneficial functions, the bacterial and fungal compositions in rhizospheres between red- and yellow-fruited tomato varieties were analyzed using high-throughput sequencing technique. Result Our results indicated that different soil microbes in rhizospheres of tomatoes were exactly recruited by different color fruit tomatoes. For the reasons as not only soil bacterial community, but also soil fungal compositions were all different between red and yellow fruit tomatoes. For example, Nocardioides , norank_f_norank_o_Vicinamibacterales , norank_f_norank_o_norank_c_KD4-96 , norank_f_Birii41 , norank_f_norank_o_S085 and Bradyrhizobium were the specific dominant soil bacterial genera, and Lecythophora , Derxomyces and unclassified_f_Pyronemataceae were the dominant soil fungal genera in the rhizospheres of red tomato varieties. By contrast, unclassified_f__Micromonsporaceae , Acidipila , Roseisolibacter , Gaiella and norank_f_Xanthobacteraceae were the unique dominant soil bacterial genera in the rhizospheres of yellow tomato varieties. And unclassified_o__Onygenales , Trichocladium , unclassified_c__Sordariomycetes , Pseudogymnoascus , Acremonium , Oidiodendron , Phialemonium , Penicillium , Phialosimplex were the unique dominant soil fungal genera in rhizospheres of yellow tomato varieties. Moreover, a higher abundance of specific soil bacterial and fungal genera in the rhizosphere was found in rhizospheres of the yellow than those of the red tomato varieties. Conclusion Soil bacterial and fungal compositions in rhizospheres between red- and yellow-fruited tomato varieties were found significantly different which growing in the same environment under the identical managements. It suggested that different soil microbes in rhizospheres exactly were recruited by different phenotypes tomato varieties related to fruit color formation.
 
Background: Mycobacterium bovis notoriously causes detrimental infections in bovines and humans. In this study, 1500 buffaloes and 2200 cattle were tested by single intradermal comparative cervical tuberculin test and compared with the detection rates of M. bovis isolation, real-time and simplex PCR, and flow Cytometry. Results: The tuberculin test is the reference test in Egypt, the positive rate was 54/3700 (1.5%) composed of 18/1500 (1.2%) buffaloes and 36/2200 (1.6%) cattle which were mandatorily slaughtered under the Egyptian legislation, after postmortem examination the non-visible-lesion proportion was 39/54 (72.2%) which surpassed the visible-lesion rate 15/54 (27.8%) with (p < 0.0001). The samples from each case were pooled into one sample representing the case, and the isolation rate of M. bovis was 25/54 (46.3%). Real-time PCR using atpE was positive for mycobacteria on the genus level in 18/18 (100%) and 5/5 (100%) of tissue samples and isolates, respectively; simplex PCR detected M. bovis in 44/54 (81.5%) and 25/25 (100%) of tissue samples and isolates, respectively. Flow Cytometry evaluation of the CD4+, CD8+, WC1+ δγ, and CD2+ cell phenotypes showed increased counts in the tuberculin-positive cases compared with negative cases (p < 0.0001), and these phenotypes in the tuberculin-positive cases increased after antigen stimulation than in the negative cases (p < 0.0001). Detection rates of PCR techniques and flow Cytometry exceeded that of bacterial isolation (p < 0.0001) and exhibited a strong correlation. Conclusions: The skin test suffers from interference from non-tuberculous mycobacteria able to cause false-positive reactions in cattle and other species. Real-time PCR using atpE, conventional PCR targeting RDs, and flow Cytometry are rapid and accurate methods that correlate with the isolation and can be promising for detection and confirmation of infected live and slaughtered cases. Keywords: Cattle and buffalo, Conventional PCR, Flow cytometry, Mycobacterium bovis, Real-time PCR atpE
 
Venn diagrams showing the number of OTUs that were unique to and shared among different communities. Rarefaction curves of the observed OTU numbers of all the samples from the six types of communities after rarefied. Percentage stacking diagrams show the community composition of bacteria and fungi at genus and species levels. The top 30 genera and species were selected for abundance analysis. Three quadrats were set for each population, with 18 quadrats in 6 populations. The abscissa is the proportion of species in the sample, and the ordinate is the plot. Different colored columns represent different species, and the length of the columns represents the proportion of the size of the species. The abbreviations of species are shown in Supplementary Table S4
NMDS analysis of species composition among the six types of communities. Different colored dots indicate different types of communities. The ellipse has a 95% confidence interval. PERMANOVA was used to evaluate intercommunity significance
Network analysis of OTU of bacteria and fungi in the six types of communities. The size of the node indicates the richness of the species. The color of the node indicates the distribution of species in different communities. A, B, C, D, E, and F were the Quercus aliena var. acutiserrata community, Quercus serrata var. brevipetiolata community, Larix gmelinii community, Pinus armandii community, Forsythia suspensa community, and Sorbus hupehensis community, respectively
Bar diagrams of bacterial and fungal distribution at the OTU level. The bar diagrams show the percent of bacteria and fungi associated with the six types of communities. Association between microbe and community was tested by torus-translation random test (Torus-translation test, P ≤ 0.05 significance level)
Variance partitioning of the effects of soil, topography, and light on the species abundance of soil bacteria (first row) and soil fungi (second row). Numbers indicate the proportions of explained variation (adjusted R² values). Values less than zero are not shown. Soil pH, soil water content, N, P, and soil organic matter. Topographical factors: elevation, slope, aspect, and convex–concave. Light: light transmittance, scattered radiation, total radiation, canopy cover, leaf area index, and average leaf angle
Background Soil microbiome is an important part of the forest ecosystem and participates in forest ecological restoration and reconstruction. Niche differentiation with respect to resources is a prominent hypothesis to account for the maintenance of species diversity in forest ecosystems. Resource-based niche differentiation has driven ecological specialization. Plants influence soil microbial diversity and distribution by affecting the soil environment. However, with the change in plant population type, whether the distribution of soil microbes is random or follows an ecologically specialized manner remains to be further studied. We characterized the soil microbiome (bacteria and fungi) in different plant populations to assess the effects of phytophysiognomy on the distribution patterns of soil microbial communities in a temperate forest in China. Results Our results showed that the distribution of most soil microbes in different types of plant populations is not random but specialized in these temperate forests. The distribution patterns of bacteria and fungi were related to the composition of plant communities. Fungal species (32%) showed higher specialization than bacterial species (15%) for different types of plant populations. Light was the main driving factor of the fungal community, and soil physicochemical factors were the main driving factor of the bacterial community. Conclusion These findings suggest that ecological specialization is important in maintaining local diversity in soil microbial communities in this forest. Fungi are more specialized than bacteria in the face of changes in plant population types. Changes in plant community composition could have important effects on soil microbial communities by potentially influencing the stability and stress resistance of forest ecosystems.
 
Background Maternally inherited bacterial symbionts are extremely widespread in insects. They owe their success to their ability to promote their own transmission through various manipulations of their hosts’ life-histories. Many symbionts however very often go undetected. Consequently, we have only a restricted idea of the true symbiont diversity in insects, which may hinder our understanding of even bigger questions in the field such as the evolution or establishment of symbiosis. Results In this study, we screened publicly available Lepidoptera genomic material for two of the most common insect endosymbionts, namely Wolbachia and Spiroplasma, in 1904 entries, encompassing 106 distinct species. We compared the performance of two screening software, Kraken2 and MetaPhlAn2, to identify the bacterial infections and using a baiting approach we reconstruct endosymbiont genome assemblies. Of the 106 species screened, 20 (19%) and nine (8.5%) were found to be infected with either Wolbachia or Spiroplasma , respectively. Construction of partial symbiotic genomes and phylogenetic analyses suggested the Wolbachia strains from the supergroup B were the most prevalent type of symbionts, while Spiroplasma infections were scarce in the Lepidoptera species screened here. Conclusions Our results indicate that many of the host-symbiont associations remain largely unexplored, with the majority of associations we identify never being recorded before. This highlights the usefulness of public databases to explore the hidden diversity of symbiotic entities, allowing the development of hypotheses regarding host-symbiont associations. The ever-expanding genomic databases provide a diverse databank from which one can characterize and explore the true diversity of symbiotic entities.
 
The FIC index of AH and EGCG on S. pyogenes (A). Effect of AH (B), EGCG (C), AH and EGCG mixture (D) on the planktonic cell growth of S. pyogenes. Data are expressed as mean SD. *P < 0.05; **P < 0.01
Effect of AH (A), EGCG (B) and AH and EGCG mixture (C) on the growth curves of S. pyogenes; effect of different concentration of AH and EGCG mixture on the growth curve of S. pyogenes coompared with AH and EGCG (D-G)
Effect of AH (A), EGCG (B) and AH and EGCG mixture (C) on the biofilm biomass of S. pyogenes analyzed by crystal violet assay. Data are expressed as mean SD. *P < 0.05; **P < 0.01
Effect of AH (A), EGCG (B) and AH and EGCG mixture (C) on the morphology of S. pyogenes by SEM observation of the biofilm after 24 h of incubation with the magnification of 1,000 × , 5,000 × , and 20,000 ×
Expression of various virulence genes in S. pyogenes after treated with AH (A), EGCG (B) and AH and EGCG mixture (C) at sub-minimum inhibitory concentration (MIC) levels compared with control group. Gene expression was quantified by RT-qPCR with 16S ribosomal RNA as the internal control. Data are expressed as mean SD. *P < 0.05; **P < 0.01. speB, streptococcal pyrogenic exotoxins B; hasA, hyaluronan synthase; luxS, S-ribosylhomocysteine lyase
Background Streptococcus pyogenes is an important global human pathogen that causes pharyngitis, and antibacterial therapy has become an important part of the overall therapy for pharyngitis. As natural derivatives, honey and green tea are often recommended for patients with pharyngitis in traditional Chinese medicine without experimental theoretical basis on wether the combined effect of honey and green tea on pharyngitis is better than they alone. The aims of this study were to explore the effects of artificial honey (AH) and epigallocatechin-3-gallate (EGCG) on S. pyogenes and elucidate the possible mechanisms, which were investigated using MIC (the minimum inhibitory concentration), FIC (fractional inhibitory concentration) index, growth pattern, biofilm formation and RT-qPCR. Results The MIC of AH on S. pyogenes was 12.5% (v/v) and the MIC of EGCG was 1250 μg/ml. The FIC index of AH and EGCG was 0.5. The planktonic cell growth, growth pattern and biofilm formation assays showed that AH and EGCG mixture had stronger inhibitory effect on S. pyogenes than they alone. RT-qPCR confirmed that the expression of hasA and luxS gene were inhibited by AH and EGCG mixture. Conclusions AH and EGCG mixture can inhibit the planktonic cell growth, biofilm formation and some virulence genes expression of S. pyogenes, better than they alone. The combination of honey and green tea have the potential to treat pharyngitis as natural derivatives, avoiding drug resistance and double infection.
 
Background The first two weeks of post-hatch (PH) growth in broilers (meat-type birds) are critical for gut development and microbiota colonization. In the current broiler production system, chicks may not receive feed and water for 24 to 72 h due to variations in hatching time and hatchery management. Post-hatch feed delay affects body weight, feed efficiency, mortality, and gut development. The goal of this study was to investigate changes in the microbiome in broiler chickens early PH and the effect of delayed access to feed on the microbiota. Results Chicks either received feed and water immediately after hatch or access to feed was delayed for 48 h to mimic commercial hatchery settings (treatment, TRT). Both groups were sampled (n = 6) at -48, 0, 4 h, and 1 (24 h), 2 (48 h), 3 (72 h), 4 (96 h), 6 (144 h), 8 (192 h), 10 (240 h), 12 (288 h) and 14 (336 h) days PH. Ileal (IL) and cecal (CE) epithelial scrapings (mucosal bacteria, M) and digesta (luminal bacteria, L) were collected for microbiota analysis. Microbiota was determined by sequencing the V3-V4 region of bacterial 16S rRNA and analyzed using QIIME2. The microbiota of early ileal and cecal samples were characterized by high abundance of unclassified bacteria. Among four bacterial populations (IL-L, IL-M, CE-L, CE-M), IL-M was the least affected by delayed access to feed early PH. Both alpha and beta diversities were affected by delayed access to feed PH in IL-L, CE-M and CE-L. However, the development effect was more pronounced. In all four bacterial populations, significant changes due to developmental effect (time relative to hatch) was observed in taxonomic composition, with transient changes of bacterial taxa during the first two weeks PH. Delayed access to feed has limited influence on bacterial composition with only a few genera and species affected in all four bacterial populations. Predicted function based on 16S rRNA was also affected by delayed access to feed PH with most changes in metabolic pathway richness observed in IL-L, CE-L and CE-M. Conclusions These results show transient changes in chicken microbiota biodiversity during the first two weeks PH and indicate that delayed access to feed affects microbiota development. Proper microbiota development could be an important factor in disease prevention and antibiotic use in broiler chickens. Moreover, significant differences in response to delayed access to feed PH between luminal and mucosal bacterial populations strongly suggests the need for separate analysis of these two populations.
 
Background Bacteria usually secrete a variety of extracellular enzymes to degrade extracellular macromolecules to meet their nutritional needs and enhance their environmental adaptability. Bacillus cereus 0–9, a biocontrol bacterial strain isolated from wheat roots, has three genes annotated as encoding amylases in the genome, but their functions are unknown, and whether they are involved in the colonization process of the bacterium remains to be further studied. Methods Mutant gene strains and fluorescently tagged strains were constructed by homologous recombination, and amylase protein was expressed in the prokaryotic Escherichia coli BL21(DE3) expression system. The iodine staining method was used to measure the activity of amylase proteins. We further observed the colonization abilities of the test strains in wheat roots through frozen section technology. Results The results showed that there were three amylase-encoding genes, amyC , amyP and amyS, in the B. cereus 0–9 genome. Among the three amylase encoding genes, only amyS produced extracellular amylase whose secretion was related to signal peptide at position 1–27. The AmyS protein encoded by the amyS gene is an α-amylase. The growth of Rhizoctonia cerealis was inhibited 84.7% by B. cereus 0–9, but the biocontrol ability of the Δ amyS strain decreased to 43.8% and that of Δ amyS / amyS was restored when the amyS gene was complemented. Furthermore, the biocontrol ability of the Δ amySec strain was decreased to 46.8%, almost the same as that of the Δ amyS mutant. Due to the deletion of the amyS gene, the colonization capacities of Δ amyS (RFP) and Δ amySec (RFP) in wheat roots decreased, while that of Δ amyS / amyS (RFP) was restored after the amyS gene was complemented, indicating that the amyS gene influences the colonization of B. cereus 0–9 in wheat roots. In addition, the colonization and biocontrol abilities of the mutant were restored after the addition of sugars, such as glucose and maltose. Conclusions B. cereus 0–9 encodes three genes annotated as amylases, amyC , amyP and amyS . Only the deletion of the amyS gene with a signal peptide did not produce extracellular amylase. The AmyS protein encoded by the amyS gene is an α-amylase. Our results indicated that the amyS gene is closely related to the colonization abilities of B. cereus 0–9 in wheat roots and the biocontrol abilities of B. cereus 0–9 to fight against R. cerealis . The extracellular amylase produced by B. cereus 0–9 can hydrolyze starch and use glucose, maltose and other nutrients to meet the needs of bacterial growth. Therefore, it is very possible that the secretion and hydrolytic activities of extracellular amylase can promote the colonization of B. cereus 0–9 in wheat roots and play important roles in the prevention and control of plant diseases. Our results contribute to exploring the mechanisms of microbial colonization in plant roots.
 
SFTSV replicates in brain tissue. A Weight changes of newborn mice after infection with SFTSV (n = 4 to 6). B Changes of viral loads in brain tissues of newborn mice infected with SFTSV (n = 4 to 6). qRT-PCR was used to detect the viral loads in brain tissues of newborn mice, and the SFTSV nucleic acid was detected at 3 hpi. The viral loads of the 3 h group were set as the benchmark, while the log2 fold changes of the other groups and the 3 h group were calculated. C Indirect immunofluorescence (IFA) detected the infectious virus particles in the brain tissues. Newborn mice were sacrificed at 3 hpi, 2 day post-infection (dpi), and 7 dpi, respectively. The brain tissues were homogenized, and the infectious SFTSV particles were detected by IFA. SFTSV was probed by SFTSV nucleoprotein (NP) human mAb and a FITC-conjugated goat anti-human IgG (H + L) secondary antibody (green); nuclei were stained with DAPI (blue). Bar = 50 μm
The target cells of SFTSV in the brain tissues of newborn mice. A colocalization of SFTSV and neurons in the brain tissues of newborn mice. a and c indicated that SFTSV infected neurons in the cerebellum; b and d indicated that SFTSV infected neurons in the midbrain. NeuN, a marker protein of mature neurons, was labeled in red. B Colocalization of SFTSV, and macrophage in brain tissues of newborn mice. a-d respectively indicated that SFTSV infected macrophages in the cerebellum, midbrain, hindbrain, and thalamus. The marker protein F4/80 of macrophages was marked in red. Nuclei were stained with DAPI (blue); SFTSV NPs were marked Green. Bar = 50 μm
Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) is an emerging tick-borne phlebovirus with a high fatality rate of 12–30%, which has an expanding endemic and caused thousands of infections every year. Central nervous system (CNS) manifestations are an important risk factor of SFTS outcome death. Further understanding of the process of how SFTSV invades the brain is critical for developing effective anti-SFTS encephalitis therapeutics. We obeserved changes of viral load in the brain at different time points after intraperitoneal infection of SFTSV in newborn C57/BL6 mice. The virus invaded the brain at 3 h post-infection (hpi). Notably, the viral load increased exponentially after 24 hpi. In addition, it was found that in addition to macrophages, SFTSV infected neurons and replicated in the brain. These findings provide insights into the CNS manifestations of severe SFTS, which may lead to drug development and encephalitis therapeutics.
 
Schematic diagram of the genetic environment of the cfr gene in this study. The arrows represent the positions and direction of the elements. The cfr gene is shown in black
Schematic diagram of the phylogenetic tree of the four strains. The strains of the research group have been marked with emphasis. The strains and their corresponding GenBank accession numbers for 16S rRNA genes are shown following the organism names. Numbers at the branching nodes are percentages of bootstrap values based on 500 replications. Bootstrap values greater than 50% are shown at the branch points. The scale bar represents 0.002 substitutions per nucleotide position
Purpose The objective of this study was to investigate the molecular characteristics and potential resistance mechanisms of linezolid-resistant (LZR) Staphylococcus capitis isolates from a tertiary hospital in China. Methods S. capitis isolates were obtained from clinical patient specimens; three of the isolates came from blood cultures and one from the hydrothorax. The agar dilution and E-test methods were used to identify antibiotic resistance. The chloramphenicol-florfenicol resistance ( cfr ) gene carrier status of the strains was determined by PCR. Whole-genome sequencing (WGS) was used to identify point mutations and L3, L4, and L22 mutations and to study the genetic environment of the cfr gene and the relationships between strains. Results The 4 isolates obtained in this study were all linezolid-resistant Staphylococcus strains. A similar of susceptibility profile pattern was observed in all four S. capitis strains, each of which exhibited a multidrug-resistant phenotype. A potentially novel mutation, C2128T, was identified, and the cfr genes of S. capitis strains were all positive. Additionally, the same mutations (C2128T and G2600T) were identified in all 23S rRNA sequences of the isolates, whereas mutations were lacking in the L3, L4, and L22 ribosomal proteins. The genetic environments surrounding cfr were identical in all four isolates. A schematic diagram of the phylogenetic tree showed that they were closely related to AYP1020, CR01, and TW2795, and a total of seven drug resistance genes were identified in these strains. Conclusions The study indicated that the resistance of the Staphylococcus capitis strains to linezolid was caused by multiple mechanisms, and a potential novel mutation, C2128T, that may have an impact on bacterial resistance was identified.
 
Background Spaceflight is a novel and profoundly stressful environment for life. One aspect of spaceflight, microgravity, has been shown to perturb animal physiology thereby posing numerous health risks, including dysregulation of normal developmental pathways. Microgravity can also negatively impact the interactions between animals and their microbiomes. However, the effects of microgravity on developmental processes influenced by beneficial microbes, such as apoptosis, remains poorly understood. Here, the binary mutualism between the bobtail squid, Euprymna scolopes, and the gram-negative bacterium, Vibrio fischeri, was studied under modeled microgravity conditions to elucidate how this unique stressor alters apoptotic cell death induced by beneficial microbes. Results Analysis of the host genome and transcriptome revealed a complex network of apoptosis genes affiliated with extrinsic/receptor-mediated and intrinsic/stress-induced apoptosis. Expression of apoptosis genes under modeled microgravity conditions occurred earlier and at high levels compared to gravity controls, in particular the expression of genes encoding initiator and executioner caspases. Functional assays of these apoptotic proteases revealed heightened activity under modeled microgravity; however, these increases could be mitigated using caspase inhibitors. Conclusions The outcomes of this study indicated that modeled microgravity alters the expression of both extrinsic and intrinsic apoptosis gene expression and that this process is mediated in part by caspases. Modeled microgravity-associated increases of caspase activity can be pharmacologically inhibited suggesting that perturbations to the normal apoptosis signaling cascade can be mitigated, which may have broader implications for maintaining animal-microbial homeostasis in spaceflight.
 
Taxonomic profiles of participant samples at phylum level. Each vertical bar represents a single participant sample. Features unassigned at phylum level were filtered prior to visualization. The brackets on the x-axis show the different age groups (A to E) and samples within each group are ordered by increasing age
The relative abundance of the top five taxa in different age groups. Taxa that could not be classified to phylum level and features appearing in less than 5 samples were filtered prior to analysis
Significant differences in fungal alpha diversity as measured by A Shannon’s H alpha diversity and B observed features, based on Kruskal–Wallis testing (p < 0.05). A: Sex (Shannon). B Vitamin A supplementation in children > 6 months (Shannon). C Antibiotic exposure < 2 weeks before sample collection (OF). D Mode of birth (OF)
A comparison of bacterial and fungal diversity in children as measured by Shannon’s H alpha diversity. After rarefaction, 5 samples were excluded from bacterial diversity analysis (n = 110), and 19 samples from the fungal analysis (n = 96)
Correlations between bacterial and fungal taxa in the gut. Red represents negative correlations and blue represents positive correlations. Only significant correlations (p < 0.05) have been coloured
Background The fungal microbiome, or mycobiome, is a poorly described component of the gut ecosystem and little is known about its structure and development in children. In South Africa, there have been no culture-independent evaluations of the child gut mycobiota. This study aimed to characterise the gut mycobiota and explore the relationships between fungi and bacteria in the gut microbiome of children from Cape Town communities. Methods Stool samples were collected from children enrolled in the TB-CHAMP clinical trial. Internal transcribed spacer 1 (ITS1) gene sequencing was performed on a total of 115 stool samples using the Illumina MiSeq platform. Differences in fungal diversity and composition in relation to demographic, clinical, and environmental factors were investigated, and correlations between fungi and previously described bacterial populations in the same samples were described. Results Taxa from the genera Candida and Saccharomyces were detected in all participants. Differential abundance analysis showed that Candida spp. were significantly more abundant in children younger than 2 years compared to older children. The gut mycobiota was less diverse than the bacterial microbiota of the same participants, consistent with the findings of other human microbiome studies. The variation in richness and evenness of fungi was substantial, even between individuals of the same age. There was significant association between vitamin A supplementation and higher fungal alpha diversity (p = 0.047), and girls were shown to have lower fungal alpha diversity (p = 0.003). Co-occurrence between several bacterial taxa and Candida albicans was observed. Conclusions The dominant fungal taxa in our study population were similar to those reported in other paediatric studies; however, it remains difficult to identify the true core gut mycobiota due to the challenges set by the low abundance of gut fungi and the lack of true gut colonising species. The connection between the microbiota, vitamin A supplementation, and growth and immunity warrants exploration, especially in populations at risk for micronutrient deficiencies. While we were able to provide insight into the gut mycobiota of young South African children, further functional studies are necessary to explain the role of the mycobiota and the correlations between bacteria and fungi in human health.
 
Varying morphology and flagella of strain EPL 0201 observed under light and electron microscopes. A Micro-curved rod-shaped bacteria (black arrow). B Micro-curved rod-shaped bacteria and flagella observed under an electron microscope
Molecular identification and virulence gene detection of strain EPL 0201. A Phylogenetic tree of Vibrio spp. based on the 16S rRNA gene sequence constructed using the neighbor-joining method. Bootstrap values were based on 1000 replicates, and percentage bootstrap values are shown at each node. B Phylogenetic tree of Vibrio spp. based on gyrB sequence constructed using the neighbor-joining method. Bootstrap values were based on 1000 replicates, and percentage bootstrap values are shown at each node. C Agarose gel electrophoresis did not show the presence of vcgC and OmpU but showed the presence of vvhA, rtxA, and Wza in the PCR products. M, Marker; Neg, negative control. Original agarose gel electropherogram (Supplementary Fig. S6)
Symptoms of fish tested for recursive infection. A Body chart of experimental fish in control group. B Abdominal cavity of fish in control group (white arrow). C Phosphorus loss in a large area of the skin (white arrow). D Hemorrhagic necrosis of the liver (aa white arrow); a large amount of fluid in the abdominal cavity (ab white arrow); and intestinal inflammation (ac white arrow). E Agarose gel electrophoresis of PCR products of vvhA and rtxA from the suspected V. vulnificus isolates. M, Marker (unit, bp); 1–4, PCR products of vvhA from V. vulnificus in samples from four diseased fishes in the 1 × 10⁷–1 × 10⁴ CFU mL⁻¹ groups; 5–8, PCR products of rtxA from V. vulnificus in samples from 4 diseased fishes in the 1 × 10⁷–1 × 10⁴ CFU mL.⁻¹ groups; 9, negative control; 10–11, positive control (PCR products of vvhA and rtxA from EPL 0201 isolate). Original agarose gel electropherogram (Fig. S6)
GC content and average depth correlation analysis chart. Note: The outermost circle represents the genome size, with each scale being 5 kb. The second circle and the third circle represent genes on the positive and negative strands of the genome. The different colors represent different COG functional classifications. The fourth circle represents a repetitive sequence, the fifth circle represents tRNA (blue) and rRNA (purple), and the sixth circle represents the GC content. The light yellow part indicates that the GC content of the region is higher than the average GC content of the genome, whereas the blue part indicates that the GC content of the region is lower than the average GC content of the genome. The higher the peak value, the greater the difference in GC content. The innermost circle represents GC skew, wherein the dark gray parts represent the regions where the G content is greater than the C content and the red parts represent the regions where the C content is greater than the G content
Species distribution map of the results of sequence alignment using the NR database. Note: Different colors represent different species
Vibrio vulnificus is a pathogenic bacterium that causes disease in marine fish, affecting fish farming and human health worldwide. In May 2021, in the Bohai Bay region, a disease broke out in commercially farmed pearl gentian grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus ), causing huge economic losses. The diseased fish had skin lesions, water accumulation in their abdomens, and showed tissue and organ damage. V. vulnificus biotype 2 has been reported in eels and other marine fish, but it is less reported in pearl gentian grouper. In this study, the pathogenic strain isolated from diseased fish was identified as V. vulnificus EPL 0201 biotype 2 on the basis of physiological and biochemical characteristics and the results of 16S rRNA gene and gyrB sequencing, virulence gene detection, and recursive infection experiments. To gain a comprehensive understanding of the pathogenicity and drug resistance of this strain, whole-genome sequencing was performed. Whole-genome analysis showed that the gene map of this strain was complete. The Virulence Factor Database annotation results showed that this strain had the key virulence factor genes vvhA and rtxA , which cause host disease. In addition, this strain had genes conferring resistance against cephalosporins, aminoglycosides, tetracyclines, and sulfonamides. Antimicrobial susceptibility testing confirmed the presence of these resistance genes identified in the genome. The results of this study show that V. vulnificus EPL 0201 biotype 2 is a multi-drug resistant strain with high pathogenicity.
 
Antimicrobial resistance of Escherichia coli from diarrheic pigs and patients. GM: gentamicin; S: streptomycin; N: neomycin; CF: cephalothin; CZ: cefazolin; FEP: cefepime; FOX: cefoxitin; NA: nalidixic acid; CIP: ciprofloxacin; NOR: norfloxacin; AMP: ampicillin; AMC: amoxicillin / clavulanic acid, SXT: trimethoprim / sulfamethoxazole; C: chloramphenicol; CL: colistin; TE: tetracycline. * Significant difference between origin of isolates (p < 0.05). ** Significant difference between origin of isolates (p < 0.01)
Multidrug resistance of E. coli from diarrheic pigs and patients in Korea. Antimicrobial subclasses defined by the Clinical and Laboratory Standards Institute (CLSI) are used. * Significant difference between origin of isolates (p < 0.05). ** Significant difference between origin of isolates (p < 0.01)
Virulence genes of E. coli from diarrheic pigs and patients in Korea. * Significant difference between origin of isolates (p < 0.05). ** Significant difference between origin of isolates (p < 0.01)
Minimum spanning tree based on sequence type of E. coli from pigs and humans
Background Escherichia ( E. ) coli causes colibacillosis in swine and humans, and is frequently associated with antimicrobial resistance. In this study we aimed to compare antimicrobial resistance, O-serogroups, virulence genes, and multi-locus sequence type of E. coli between isolates from pigs and patients suffering from diarrhea, and the most prevalent pathogenic E. coli strain from swine isolates in Korea. Methods We tested 64 and 50 E. coli strains from pigs and patients suffering from diarrhea for antimicrobial susceptibility test, virulence genes, O-serogroups, and multi-locus sequence typing. Results We confirmed that isolates from swine showed significantly higher resistance than from those from patients, especially to fluoroquinolone (ciprofloxacin: 37.5 and 10.0%; norfloxacin: 29.7 and 8.0%, respectively). Stx1 (46.0%) was most frequently detected in patients followed by stx2 (38.0%). There was no significant difference in stx2 (swine: 23.4%, patients: 38.0%). In isolates from patients, O157 (12.0%) was the most prevalent O-serogroup, and two isolates (3.1%) from pigs were confirmed to have O157. Additionally, sequence type (ST) 10 (swine: 6 isolates, patients: 2 isolates) and ST 88 (swine: 2 isolates, patients: 1 isolate) were simultaneously detected. Conclusions We found that both isolates from swine and human had the stx2 gene, which could cause severe disease. Moreover, antimicrobial resistance was significantly higher in pigs than in patients. These results suggest that pig could act as a reservoir in human infection and antimicrobial resistance could be transferred to human from pigs.
 
Background Acinetobacter baumannii has emerged as the major opportunistic pathogen in healthcare-associated infections with high-level antibiotic resistance and high mortality. Quorum sensing (QS) system is a cell-to-cell bacterial communication mediated by the synthesis, secretion, and binding of auto-inducer signals. It is a global regulatory system to coordinate the behavior of individual bacteria in a population. The present study focused on the QS system, aiming to investigate the regulatory role of QS in bacterial virulence and antibiotic resistance. Method The auto-inducer synthase gene abaI was deleted using the A. baumannii ATCC 19606 strain to interrupt the QS process. The RNA-seq was performed to identify the differentially expressed genes (DEGs) and pathways in the mutant (△abaI) strain compared with the wild-type (WT) strain. Results A total of 380 DEGs [the adjusted P value < 0.05 and the absolute value of log 2 (fold change) > log 2 1.5] were identified, including 256 upregulated genes and 124 downregulated genes in the △abaI strain. The enrichment analysis indicated that the DEGs involved in arginine biosynthesis, purine metabolism, biofilm formation, and type VI secretion system (T6SS) were downregulated, while the DEGs involved in pathways related to fatty acid metabolism and amino acid metabolism were upregulated. Consistent with the expression change of the DEGs, a decrease in biofilm formation was observed in the △abaI strain compared with the WT strain. On the contrary, no obvious changes were found in antimicrobial resistance following the deletion of abaI . Conclusions The present study demonstrated the transcriptomic profile of A. baumannii after the deletion of abaI , revealing an important regulatory role of the QS system in bacterial virulence. The deletion of abaI suppressed the biofilm formation in A. baumannii ATCC 19606, leading to decreased pathogenicity. Further studies on the role of abaR , encoding the receptor of auto-inducer in the QS circuit, are required for a better understanding of the regulation of bacterial virulence and pathogenicity in the QS network.
 
Background The aging process in the tobacco production, as in other food industries, is an important process for improving the quality of raw materials. In the spontaneous aging, the complex components in flue-cured tobacco (FT) improve flavor or reduce harmful compounds through chemical reactions, microbial metabolism, and enzymatic catalysis. Some believed that tobacco-microbe played a significant part in this process. However, little information is available on how microbes mediate chemical composition to improve the quality of FT, which will lay the foundation for the time-consuming spontaneous aging to seek ways to shorten the aging cycle. Results Comparing aged and unaged FT, volatile and non-volatile differential compounds (DCs) were multi-dimensionally analyzed with the non-targeted metabolomes based on UPLC-QTOP-MS (the ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry), GC–MS (gas chromatography-mass spectrometer) assisted derivatization and HP-SPME-GC/MS (headspace solid-phase micro-extraction assisted GC–MS). Products associated with the degradation pathways of terpenoids or higher fatty acids were one of the most important factors in improving FT quality. With the microbiome, the diversity and functions of microbial flora were analyzed. The high relative abundance function categories were in coincidence with DCs-related metabolic pathways. According to the correlation analysis, Acinetobacter , Sphingomonas and Aspergillus were presumed to be the important contributor, in which Aspergillus was associated with the highest number of degradation products of terpenoids and higher fatty acids. At last, the screened Aspergillus nidulans strain F4 could promote the degradation of terpenoids and higher fatty acids to enhance tobacco flavor by secreting highly active lipoxygenase and peroxidase, which verified the effect of tobacco-microbes on FT quality. Conclusions By integrating the microbiome and metabolome, tobacco-microbe can mediate flavor-related substances to improve the quality of FT after aging, which provided a basis for identifying functional microorganisms for reforming the traditional spontaneous aging.
 
Minimum inhibitory concentrations (MICs) distribution of clarithromycin (A), levofloxacin (B), metronidazole (C), amoxicillin (D), tetracycline (E), and rifampicin (F) to 153 H. pylori isolates. Dashed arrows indicate the resistance breakpoints with MIC > 0.5 mg/L for clarithromycin, MIC > 1 mg/L for levofloxacin, MIC > 8 mg/L for metronidazole, MIC > 0.125 mg/L for amoxicillin, MIC > 1 mg/L for tetracycline and MIC > 1 mg/L for rifampicin. S, Sensitive; R, Resistant
The overall sensitive and resistance rates of the 153 H. pylori isolates against seven antibiotics. The resistance to clarithromycin, levofloxacin, metronidazole, amoxicillin, tetracycline, rifampicin was determined by E-test, and the resistance to furazolidone was determined by disk diffusion method
Background The prevalence of Helicobacter pylori antibiotic susceptibility in the Tibet Autonomous Region, China is not determined. This study aimed to evaluate the antibiotic resistance patterns of H. pylori isolates there. Results A total of 153 (38.5%) H. pylori strains were successfully isolated from 397 patients in People's Hospital of Tibet Autonomous Region, China. The overall resistance rates were as follows: clarithromycin (27.4%), levofloxacin (31.3%), metronidazole (86.2%), amoxicillin (15.6%), tetracycline (0%), furazolidone (0.6%), and rifampicin (73.2%). Only 2.0% of H. pylori isolates were susceptible to all tested antimicrobials, with mono resistance, dual resistance, triple resistance, quadruple resistance, and quintuple resistance being 18.3%, 44.4%, 18.3%, 12.4%, and 4.6%, respectively. The resistance rates to levofloxacin (40.5%) and amoxicillin (21.5%) in strains isolated from female patients were significantly higher than those from male patients (21.6% and 9.5%, respectively). Conclusions This study demonstrates high H. pylori resistance rates to clarithromycin, levofloxacin, metronidazole, and rifampicin, whereas moderate resistance to amoxicillin, and negligible resistant to tetracycline, and furazolidone in Tibet Autonomous Region, China. The high resistance to rifampicin warns further investigation of its derivative, rifabutin.
 
A X-ray diffraction pattern of zinc oxide nanoparticles. B TEM image of the rectangular shaped zinc oxide nanoparticles
Hoechst H33342 accumulation assay in E.coli isolate (#1063) in the presence and absence of CCCP and different nanoparticles (Ag, CU and ZnO). Heat-inactivated bacteria served as a positive control. The fluorescence intensity was recorded at excitation and emission wavelengths of 360 nm and 460 nm, respectively, over 80 min incubation period
Hoechst H33258 accumulation in enterococcal isolate (#B59) in the presence and absence of CCCP and different nanoparticles (Ag, CU and ZnO). The fluorescence intensity was recorded at excitation and emission wavelengths of 360 nm and 460 nm, respectively, over 120 min incubation period
A, B, C Fold differences in levels of H33258 accumulated by enterococcal isolates at steady- state with or without efflux inhibitors (CCCP and ZnONPs). (*) Significant difference in dye accumulation in the presence of efflux inhibitors in comparison with the lack of them. P-value of ≤ 0.05 was considered significant. Each experiment was repeated three times
Background Contribution of efflux pumps in development of antimicrobial resistance has been largely addressed in Gram negative and to a much lesser extent in Gram positive bacteria. Measuring accumulation of Hoechst (H) dye is known as a safe and rapid method for monitoring efflux activity in bacteria. Antimicrobial effects of metal nanoparticles have been attributed in part to inhibition of efflux pumps. This study aimed to first determine efflux activity in enterococci by Hoechst accumulation assay, and to second characterize the role of zinc oxide nanoparticles (ZnONPs) in inhibition of these pumps. Results Increased accumulation of Hoechst dye showed more potential of ZnONPs in efflux inhibition compared with CCCP. H33258 represented more suitability for accumulation studies in enterococci. Two to six-fold reduction in minimum inhibitory concentration (MIC) values of antimicrobial agents in the presence of ZnONPs was observed. Conclusions Efflux activity in enterococcal strains can be measured by H33258 accumulation assay. Application of ZnONPs as an efflux inhibitor, may rejuvenate the use of conventional antimicrobial agents against these bacteria.
 
Background Aliivibrio salmonicida is the causative agent of cold-water vibriosis in salmonids ( Oncorhynchus mykiss and Salmo salar L.) and gadidae ( Gadus morhua L.). Virulence-associated factors that are essential for the full spectrum of A. salmonicida pathogenicity are largely unknown. Chitin-active lytic polysaccharide monooxygenases (LPMOs) have been indicated to play roles in both chitin degradation and virulence in a variety of pathogenic bacteria but are largely unexplored in this context. Results In the present study we investigated the role of LPMOs in the pathogenicity of A. salmonicida LFI238 in Atlantic salmon ( Salmo salar L.). In vivo challenge experiments using isogenic deletion mutants of the two LPMOs encoding genes AsLPMO10A and AsLPMO10B, showed that both LPMOs, and in particular AsLPMO10B, were important in the invasive phase of cold-water vibriosis. Crystallographic analysis of the AsLPMO10B AA10 LPMO domain (to 1.4 Å resolution) revealed high structural similarity to viral fusolin, an LPMO known to enhance the virulence of insecticidal agents. Finally, exposure to Atlantic salmon serum resulted in substantial proteome re-organization of the A. salmonicida LPMO deletion variants compared to the wild type strain, indicating the struggle of the bacterium to adapt to the host immune components in the absence of the LPMOs. Conclusion The present study consolidates the role of LPMOs in virulence and demonstrates that such enzymes may have more than one function.
 
Background Plant microbiome and its manipulation inaugurate a new era for plant biotechnology with the potential to benefit sustainable crop production. Here, we used the large-scale 16S rDNA sequencing analysis to unravel the dynamic, structure, and composition of exophytic and endophytic microbial communities in two hybrid commercial cultivars of sugarcane (R570 and SP80–3280), two cultivated genotypes ( Saccharum officinarum and Saccharum barberi ) and one wild species ( Saccharum spontaneum ). Results Our analysis identified 1372 amplicon sequence variants (ASVs). The microbial communities’ profiles are grouped by two, root and bulk soils and stem and leave when these four components are compared. However, PCoA-based data supports that endophytes and epiphytes communities form distinct groups, revealing an active host-derived mechanism to select the resident microbiota. A strong genotype-influence on the assembly of microbial communities in Saccharum ssp. is documented. A total of 220 ASVs persisted across plant cultivars and species. The ubiquitous bacteria are two potential beneficial bacteria, Acinetobacter ssp., and Serratia symbiotica . Conclusions The results presented support the existence of common and cultivar-specific ASVs in two commercial hybrids, two cultivated canes and one species of Saccharum across tissues (leaves, stems, and roots). Also, evidence is provided that under the experimental conditions described here, each genotype bears its microbial community with little impact from the soil conditions, except in the root system. It remains to be demonstrated which aspect, genotype, environment or both, has the most significant impact on the microbial selection in sugarcane fields.
 
Background: Bacterial biofilm can occur on all medical implanted devices and lead to infection and/or dysfunction of the device. In this study, artificial biofilm was formed on four different medical implants (silicone, piccline, peripheral venous catheter and endotracheal tube) of interest for our daily clinical and/or research practice. We investigated the best conventional technic to dislodge the biofilm on the implants and quantified the number of bacteria. Staphylococcus epidermidis previously isolated from a breast implant capsular contracture on a patient in the university hospital of Dijon was selected for its ability to produce biofilm on the implants. Different technics (sonication, Digest-EUR®, mechanized bead mill, combination of sonication plus Digest-EUR®) were tested and compared to detach the biofilm before quantifying viable bacteria by colony counting. Results: For all treatments, the optical and scanning electron microscope images showed substantial less biofilm biomass remaining on the silicone implant compared to non-treated implant. This study demonstrated that the US procedure was statistically superior to the other physical treatment: beads, Digest-EUR® alone and Digest-EUR® + US (p < 0.001) for the flexible materials (picc-line, PIV, and silicone). The number of bacteria released by the US is significantly higher with a difference of 1 log on each material. The result for a rigid endotracheal tube were different with superiority for the chemical treatment dithiothreitol: Digest-EUR®. Surprisingly the combination of the US plus Digest-EUR® treatment was consistently inferior for the four materials. Conclusions: Depending on the materials used, the biofilm dislodging technique must be adapted. The US procedure was the best technic to dislodge S. epidermidis biofilm on silicone, piccline, peripheral venous catheter but not endotracheal tube. This suggested that scientists should compare themselves different methods before designing a protocol of biofilm study on a given material.
 
Background Huperzine A (Hup A) has attracted considerable attention as an effective therapeutic candidate drug used to treat Alzheimer’s disease. Whereas, the production of Hup A from wild plants faced a major challenge, which is the wild Huperzia Serrata harbor a low Hup A content, has a long-life cycle, and has a small yield. At present, several reports showed that Hup A is produced by various endophytic fungal strains isolated from H. serrata , thereby providing an alternative method to produce the compound and reduce the consumption of this rare and endangered plant. However, till now, very few comprehensive studies are available on the biological diversity and structural composition of endophytic fungi and the effects of endophytic fungi on the Hup A accumulation in H. serrata . Results In this research, the composition and diversity of fungal communities in H. serrata were deciphered based on high-throughput sequencing technology of fungal internal transcribed spacer regions2 (ITS2). The correlation between endophytic fungal community and Hup A content was also investigated. Results revealed that the richness and the diversity of endophytic fungi in H. serrata was various according to different tissues and different ecological areas. The endophytic fungal communities of H. serrata exhibit species-specific, ecological-specific, and tissue-specific characteristics. There are 6 genera (Ascomycota_unclassified, Cyphellophora, Fungi_unclassified, Sporobolomyces, and Trichomeriaceae_unclassified) were significantly positively correlated with Hup A content in all two areas, whereas, there are 6 genera ( Auricularia, Cladophialophora, Cryptococcus, Mortierella, and Mycena ) were significantly negatively correlated with Hup A content of in all two areas. Conclusions This study indicated a different composition and diverse endophytic fungal communities in H. serrata from different organs and ecological areas. The current study will provide the realistic basis and theoretical significance for understanding the biological diversity and structural composition of endophytic fungal communities in H. serrata , as well as providing novel insights into the interaction between endophytic fungi and Hup A content.
 
Structure and sequence of the putative cell-surface hydrolase, CshA. Schematic diagram of the CshA, and its amino acid sequence (A). The sequence for the signal peptide (SP) is underlined, while the sequence for the active site containing the alpha/beta hydrolase is in bold. Three-dimensional structure of CshA rendered by I-TASSER showing the alternating α-helices and β-strands (B)
Expression of the sfGFP and CshA-sfGFP proteins. Overexpression of the sfGFP (28 kDa) and CshA-sfGFP (63 kDa) was confirmed through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (A), and Western blot (B). (Gels and blots were cropped for clarity. The full-length images are included in the Additional file, Figure S3)
Localization of the CshA-mediated surface display of sfGFP on LAB. Surface display of CshA on Lactiplantibacillus plantarum SK151, Lp. plantarum SK156, Limosilactobacillus fermentum SK152, Lm. mucosae LM1, and Lactobacillus johnsonii PF01 (A). Cell-associated fluorescence was observed using fluorescence microscopy (top row). sfGFP alone cannot bind to the cell surface of Lm. fermentum (B). Surface binding of CshA to LAB is host-dependent, with preference to Lm. fermentum (C). Significant differences were determined using ANOVA with Tukey’s test for pairwise comparison of means and denoted by difference in letters
Optimization of the CshA binding on Lm. fermentum cells. Pretreatment of the Lm. fermentum cells affected the surface binding of CshA (A). NaCl concentration (B) and pH (C), as well as the binding time and temperature (D) also influenced the surface binding of CshA. The binding capacity of pretreated Lm. fermentum cells was determined by fitting the fluorescence at different protein concentration into a nonlinear curve (E), then calculated using a standard curve (F). All experiments were done in triplicates and reported as mean ± SD. Significant differences were determined using ANOVA with Tukey’s test for pairwise comparison of means. Differences are denoted by ** P < 0.01 and *** P < 0.001, or by different letters
CshA-mediated surface display on pretreated Lm. fermentum showed stability under simulated gastrointestinal tract conditions. There was no significant (ns) loss of fluorescence at different pH (3–5) and bile concentrations (0.25%–1%) compared with the control setup. All experiments were done in triplicates and reported as mean ± SD. Significant differences were determined using ANOVA with Tukey’s test for pairwise comparison of means
Recent studies have demonstrated the potential of surface display technology in therapeutic development and enzyme immobilization. Utilization of lactic acid bacteria in non-GMO surface display applications is advantageous due to its GRAS status. This study aimed to develop a novel, non-GMO cell wall anchoring system for lactic acid bacteria using a cell-surface hydrolase (CshA) from Lactiplantibacillus plantarum SK156 for potential industrial and biomedical applications. Analysis of the CshA revealed that it does not contain any known classical anchor domains. Although CshA lacks a classical anchor domain, it successfully displayed the reporter protein superfolder GFP on the surface of several lactic acid bacteria in host dependent manner. CshA-sfGFP fusion protein was displayed greatest on Limosilactobacillus fermentum SK152. Pretreatment with trichloroacetic acid further enhanced the binding of CshA to Lm. fermentum . The binding conditions of CshA on pretreated Lm. fermentum (NaCl, pH, time, and temperature) were also optimized, resulting in a maximum binding of up to 10 ⁶ CshA molecules per pretreated Lm. fermentum cell. Finally, this study demonstrated that CshA-decorated pretreated Lm. fermentum cells tolerates gastrointestinal stress, such as low pH and presence of bile acid. To our knowledge, this study is the first to characterize and demonstrate the cell-surface display ability of CshA. The potential application of CshA in non-GMO antigen delivery system and enzyme immobilization remains to be tested.
 
Despite the known influence of continuous cropping on soil microorganisms, little is known about the associated difference in the effects of continuous cropping on the community compositions of soil bacteria and fungi. Here, we assessed soil physicochemical property, as well as bacterial and fungal compositions across different years (Uncropped control, 1, 6, 11, 16, and 21 years) and in the watermelon system of a gravel mulch field in the Loess Plateau of China. Our results showed that long-term continuous cropping led to substantial shifts in soil bacterial and fungal compositions. The relative abundances of dominant bacterial and fungal genera (average relative abundance > 1.0%) significantly varied among different continuous cropping years (P < 0.05). Structural equation models demonstrated that continuous cropping alter soil bacterial and fungal compositions mainly by causing substantial variations in soil attributes. Variations in soil pH, nutrient, salinity, and moisture content jointly explained 73% and 64% of the variation in soil bacterial and fungal compositions, respectively. Variations in soil moisture content and pH caused by continuous cropping drove the shifts in soil bacterial and fungal compositions, respectively (Mantel R = 0.74 and 0.54, P < 0.01). Furthermore, the variation in soil bacterial and fungal composition showed significant correlation with watermelon yield reduction (P < 0.01). Together, long-term continuous cropping can alter soil microbial composition, and thereby influencing watermelon yield. Our findings are useful for alleviating continuous cropping obstacles and guiding agricultural production.
 
Background Pseudomonas species are widely distributed in the human body, animals, plants, soil, fresh water, seawater, etc. Pseudomonas aeruginosa is one of the main pathogens involved in nosocomial infections. It can cause endocarditis, empyema, meningitis, septicaemia and even death. However, the Pseudomonas classification system is currently inadequate and not well established. Results In this study, the whole genomes of 103 Pseudomonas strains belonging to 62 species available in GenBank were collected and the specificity of the 16S–23S ribosomal RNA internal transcribed spacer (ITS) sequence was analysed. Secondary structures of ITS transcripts determining where the diversity bases were located were predicted. The alignment results using BLAST indicated that the ITS sequence is specific for most species in the genus. The remaining species were identified by additional frequency analyses based on BLAST results. A double-blind experiment where 200 ITS sequences were randomly selected indicated that this method could identify Pseudomonas species with 100% sensitivity and specificity. In addition, we applied a universal primer to amplify the Pseudomonas ITS of DNA extracts from fish samples with next-generation sequencing. The ITS analysis results were utilized to species-specifically identify the proportion of Pseudomonas species in the samples. Conclusions The present study developed a species-specific method identification and classification of Pseudomonas based on ITS sequences combined NGS. The method showed its potential application in other genera.
 
Pathogenicity of Pseudomonas aeruginosa AB030 in mice models and efficacy of Pseudomonas phage AP025 (A) and AP006 (B) against P. aeruginosa bacteremia infections. Representative survival curves of mice following infection by clinical P. aeruginosa strain AB030 and treated with phages 2 hours post-infection. The mice in the infected group were dead within 48 hours. In the phage control group, 100% survival was observed. When infected mice were treated with phage AP025 at MOI = 1, 1/6 mice were dead on the 2nd day. With phage AP006 at MOI = 1, we observed death in 2 of 6 mice on the 1st and the 2nd day. All the animals survived when treated with phages at MOI values of 10 and 100, respectively, with signs of infection. The survival curves were plotted using the Kaplan-Meier method and the log-rank test was used to analyze the difference in survival rates in GraphPad Prism 7.0. A statistically significant difference (p < 0.05) was observed in the treatment groups
Bacterial enumeration from mice post-infection with P. aeruginosa and treated with Pseudomonas phage AB025 (MOI = 10) and AB006 (MOI = 10). Results are represented as CFU for bacteria isolated from different organs in the mice after 48 hours. Error bars represent the standard error of the mean (SEM) of three independent replicates. Data were analyzed using GraphPad Prism 7.0 and two-way ANOVA was used for statistical analysis (P < 0.05 was considered significant)
Background Pseudomonas aeruginosa is an opportunistic pathogen that can cause a variety of infections in humans, such as burn wound infections and infections of the lungs, the bloodstream and surgical site infections. Nosocomial spread is often concurrent with high degrees of antibiotic resistance. Such resistant strains are difficult to treat, and in some cases, even reserved antibiotics are ineffective. A particularly promising therapy to combat infections of resistant bacteria is the deployment of bacteriophages, known as phage therapy. In this work, we evaluated the in vivo efficacy of two Pseudomonas phages in bacteremia mice models. For this study, non-neutropenic mice (BalB/C) were infected with P. aeruginosa AB030 strain and treated using two bacteriophages, AP025 and AP006. Results The results showed that a single dose of phages at higher concentrations, bacteria: phage at 1:10 and 1:100 were effective in eliminating the bloodstream infection and achieving 100% mice survival. Conclusion This study highlights the efficacy of using a single dose of phages to restore mice from bacteremia.
 
Background Cellulolytic microorganisms are considered a key player in the degradation of feed fiber. These microorganisms can be isolated from various resources, such as animal gut, plant surfaces, soil and oceans. A new strain of Bacillus amyloliquefaciens , TL106, was isolated from faeces of a healthy Tibetan pigs. This strain can produce cellulase and shows strong antimicrobial activity in mice. Thus, in this study, to better understand the strain of B. amyloliquefaciens TL106 on degradation of cellulose, the genome of the strain TL106 was completely sequenced and analyzed. In addition, we also explored the cellulose degradation ability of strain TL106 in vitro. Results TL106 was completely sequenced with the third generation high-throughput DNA sequencing. In vitro analysis with enzymatic hydrolysis identified the activity of cellulose degradation. TL106 consisted of one circular chromosome with 3,980,960 bp and one plasmid with 16,916 bp, the genome total length was 3.99 Mb and total of 4,130 genes were predicted. Several genes of cellulases and hemicellulase were blasted in Genbank, including β-glucosidase, endoglucanase, ß-glucanase and xylanase genes. Additionally, the activities of amylase (20.25 U/mL), cellulase (20.86 U/mL), xylanase (39.71 U/mL) and β-glucanase (36.13 U/mL) in the fermentation supernatant of strain TL106 were higher. In the study of degradation characteristics, we found that strain TL106 had a better degradation effect on crude fiber, neutral detergent fiber, acid detergent fiber, starch, arabinoxylan and β-glucan of wheat and highland barley . Conclusions The genome of B. amyloliquefaciens TL106 contained several genes of cellulases and hemicellulases, can produce carbohydrate-active enzymes, amylase, cellulase, xylanase and β-glucanase. The supernatant of fermented had activities of strain TL106. It could degrade the fiber fraction and non-starch polysaccharides (arabinoxylans and β-glucan) of wheat and highland barley. The present study demonstrated that the degradation activity of TL106 to crude fiber which can potentially be applied as a feed additive to potentiate the digestion of plant feed by monogastric animals.
 
Background β-glucosidase is an important biomass-degrading enzyme and plays a vital role in generating renewable biofuels through enzymatic saccharification. In this study, we analyzed the transcriptome of Trichoderma harzianum HTASA derived from Hainan mangrove and identified a new gene encoding β-glucosidase Bgl3HB. And the biochemically characterization of β-glucosidase activity was performed. Results Bgl3HB showed substantial catalytic activity in the pH range of 3.0–5.0 and at temperatures of 40 ℃-60 ℃. The enzyme was found quite stable at 50 ℃ with a loss of only 33.4% relative activity after 240 min of heat exposure. In addition, all tested metal ions were found to promote the enzyme activity. The β-glucosidase activity of Bgl3HB was enhanced by 2.12-fold of its original activity in the presence of 5 M NaCl. Surprisingly, Bgl3HB also showed a remarkable ability to hydrolyze laminarin compared to other measured substrates. Enzyme efficiency was examined in the sugarcane bagasse saccharification processes, in which Bgl3HB with 5 M NaCl worked better supplementing Celluclast 1.5L than the commercial Novozyme 188 ascertained it as an admirably suited biocatalyst for the utilization of agricultural waste. In this work, this is the first report of a halophilic β-glucosidase from Trichoderma harzianum , and represents the β-glucosidase with the highest known NaCl activation concentration. And adding 5 M NaCl could enhance saccharification performance even better than commercial cellulase. Conclusions These results show that Bgl3HB has great promise as a highly stable and highly efficient cellulase with important future applications in the industrial production of biofuels.
 
A DESeq2 identifies specific bacterial taxa associated with the development of GC, with age, gender, H. pylori, and batch effects adjusted. B NetMoss2 identifies specific bacterial taxa in microbial Sparcc networks between GC and SG. C Sparcc networks between GC and SG constructed by NetMoss2. HC: health control, SG: superficial gastritis, AG: atrophic gastritis, IM: intestinal metaplasia, IN: intraepithelial neoplasia, GC: gastric cancer, CAN: carcinoma adjacent normal tissues. Log2(FC): Log2(Fold Change), -loge(P): the negative of log base e of P value
Differences of microbial composition and diversity between three bacterial communities. A, B The multidimensional cluster analysis of the gastric microbiome at the genus level shows three distinct clusters of samples. C The abundance of the gastric microbiota is shown for phylum level in three microbial communities. D Alpha diversity was estimated by the Shannon index at the genus level for three microbial communities. E The relative abundance of significantly changed bacteria in GT-F type samples among different disease groups. GT: gastric type. *: p.adj < 0.05, **: p.adj < 0.01, ***: p.adj < 0.001
Gastric microbial diversity and community structure at the genus level among disease groups. A Alpha diversity was estimated by Shannon diversity index for disease groups. B PCoA plots and PERMANOVA test based on Bray–Curtis distance for disease groups. HC: health control, SG: superficial gastritis, AG: atrophic gastritis, IM: intestinal metaplasia, IN: intraepithelial neoplasia, GC: gastric cancer, CAN: carcinoma adjacent normal tissues. *: adjusted p < 0.05, **: adjusted p < 0.01, ***: adjusted p < 0.001
Correlation networks of the gastrointestinal genus among disease groups. A The interactions of bacteria in gastric biopsies in samples of GT-H. B The interactions of bacteria in samples of GT-F. C The interactions of bacteria in samples of GT-P. The size of nodes corresponds to weighted node connectivity scores, and the nodes were colored by phylum. Red edges denote positive correlations and blue edges denote negative correlations. The interactions of bacteria in GC and CAN groups were exhibited with strengths > 0.4, and in SG, AG, IM, IN groups with strengths > 0.6. HC: health control, SG: superficial gastritis, AG: atrophic gastritis, IM: intestinal metaplasia, IN: intraepithelial neoplasia, GC: gastric cancer, CAN: carcinoma adjacent normal tissues
Based on genera selected by Boruta, the performance of gastric microbial models in prediction GC was analysed by receiver operating characteristic (ROC) curve analysis. A The discriminatory potential of microbial model in distinguishing GC and SG. B The discriminatory potential of microbial model in distinguishing GC and precancerous lesions, including AG, IM, and IN. C The discriminatory potential of microbial model in distinguishing GC and CAN. SG: superficial gastritis, IM: intestinal metaplasia, GC: gastric cancer, CAN: carcinoma adjacent normal tissues, preGC: precancerous lesions of gastric cancer. AUC: area under the curve
Background Gastric microbial dysbiosis were reported to be associated with gastric cancer (GC). This study aimed to explore the variation, diversity, and composition patterns of gastric bacteria in stages of gastric carcinogenesis based on the published datasets. Methods We conducted a gastric microbial analysis using 10 public datasets based on 16S rRNA sequencing, including 1270 gastric biopsies of 109 health control, 183 superficial gastritis (SG), 135 atrophic gastritis (AG), 124 intestinal metaplasia (IM), 94 intraepithelial neoplasia (IN), 344 GC, and 281 adjacent normal tissues. And QIIME2-pipeline, DESeq2, NetMoss2, vegan, igraph, and RandomForest were used for the data processing and analysis. Results We identified three gastric microbial communities among all the gastric tissues. The first community (designate as GT-H) was featured by the high abundance of Helicobacter . The other two microbial communities, namely GT-F, and GT-P, were featured by the enrichment of phylum Firmicutes and Proteobacteria, respectively. The distribution of GC-associated bacteria, such as Fusobacterium , Peptostreptococcus , Streptococcus , and Veillonella were enriched in tumor tissues, and mainly distributed in GT-F type microbial communities. Compared with SG, AG, and IM, the bacterial diversity in GC was significantly reduced. And the strength of microbial interaction networks was initially increased in IM but gradually decreased from IN to GC. In addition, Randomforest models constructed in in GT-H and GT-F microbial communities showed excellent performance in distinguishing GC from SG and precancerous stages, with varied donated bacteria. Conclusions This study identified three types of gastric microbiome with different patterns of composition which helps to clarify the potential key bacteria in the development of gastric carcinogenesis.
 
To study the mechanism of lactoferrin (LF) regulating metabolic disorders in nutritionally obese mice through intestinal microflora. Twenty-one male C57BL/6 mice were randomly divided into 3 groups: control group, model group and LF treatment group. The mice in control group were fed with maintenance diet and drank freely. The mice in model group were fed with high fat diet and drank freely. The mice in LF treatment group were fed with high fat diet and drinking water containing 2% LF freely. Body weight was recorded every week. Visceral fat ratio was measured at week 12. Blood glucose and serum lipid level were detected by automatic biochemical analyzer. The gut microbiota of mice was examined using 16 s rRNA sequencing method. LF treatment significantly reduced the levels of visceral adipose ratio, blood glucose, triglyceride, total cholesterol and low-density lipoprotein cholesterol (LDL-C) in high-fat diet mice (p < 0.05). It can be seen that drinking water with 2% LF had a significant impact on metabolic disorders. At the same time, the Firmicutes/Bacteroidetes ratio(F/B) of LF treated mice was decreased. The abundance of Deferribacteres, Oscillibacter, Butyricicoccus, Acinetobacter and Mucispirillum in LF treatment group were significantly decreased, and the abundance of Dubosiella was significantly increased (p < 0.05). In the LF-treated group, the expression levels of glucose metabolism genes in gut microbiota were increased, and the expression levels of pyruvate metabolism genes were decreased. It can be seen that metabolic disorders were related to intestinal flora. In conclusion, LF regulates metabolic disorders by regulating intestinal flora.
 
Background Polyhydroxybutyrate (PHB) is a biopolymer formed by some microbes in response to excess carbon sources or essential nutrient depletion. PHBs are entirely biodegradable into CO2 and H2O under aerobic and anaerobic conditions. It has several applications in various fields such as medicine, pharmacy, agriculture, and food packaging due to its biocompatibility and nontoxicity nature. Result In the present study, PHB-producing bacterium was isolated from the Dirout channel at Assiut Governorate. This isolate was characterized phenotypically and genetically as Bacillus cereus SH-02 (OM992297). According to one-way ANOVA test, the maximum PHB content was observed after 72 h of incubation at 35 °C using glucose and peptone as carbon and nitrogen source. Response surface methodology (RSM) was used to study the interactive effects of glucose concentration, peptone concentration, and pH on PHB production. This result proved that all variables have a significant effect on PHB production either independently or in the interaction with each other. The optimized medium conditions with the constraint to maximize PHB content and concentration were 22.315 g/L glucose, and 15.625 g/L peptone at pH 7.048. The maximum PHB content and concentration were 3100.799 mg/L and 28.799% which was close to the actual value (3051 mg/l and 28.7%). The polymer was identified as PHB using FTIR, NMR, and mass spectrometry. FT-IR analysis showed a strong band at 1724 cm− 1 which attributed to the ester group’s carbonyl while NMR analysis has different peaks at 169.15, 67.6, 40.77, and 19.75 ppm that were corresponding to carbonyl, methine, methylene, and methyl resonance. Mass spectroscopy exhibited molecular weight for methyl 3- hydroxybutyric acid. Conclusion PHB–producing strain was identified as Bacillus cereus SH-02 (OM992297). Under optimum conditions from RSM analysis, the maximum PHB content and concentration of this strain can reach (3100.799 mg/L and 28.799%); respectively. FTIR, NMR, and Mass spectrometry were used to confirm the polymer as PHB. Our results demonstrated that optimization using RSM is one of the strategies used for reducing the production cost. RSM can determine the optimal factors to produce the polymer in a better way and in a larger quantity without consuming time.
 
Background Long-term cultivation of ginseng can cause severe crop disorders and soil sickness. Crop rotation is an effective agricultural management measure to improve soil sustainability and decrease pathogens. However, the suitable ginseng rotation system and the changes in soil microbial community and soil characteristics under the rotation system need to be further explored. Methods To explore suitable ginseng crop rotation systems and improve soil utilization, Allium fistulosum and Brassica napus were planted on ginseng cultivation soil for one year. The effects of the two crops on the chemical properties and enzyme activities of the ginseng cultivation soil were evaluated by chemical analysis. In addition, amplicon sequencing targeting 16 s rDNA genes of bacteria and ITS of fungi has been used to characterize the functional and compositional diversity of microbial communities. Results The results elucidated that the levels of available phosphorus (AP) and available potassium (AK) in the soil increased significantly after one year of cultivation for both crops and Allium fistulosum cultivation may also have reduced soil salinity. In addition, the effects of the two crops on the activities of key soil enzymes were different. Catalase (CAT), urease (URE), and acid phosphatase (A-PHO) activities were significantly reduced and sucrase (SUC), and laccase (LAC) activities were significantly increased after Allium fistulosum planting. While A-PHO activity was significantly increased and LAC activity was significantly decreased after Brassica napus planting. Allium fistulosum significantly reduced the abundance of soil fungal communities. The cultivation of Allium fistulosum and Brassica napus significantly altered the composition of soil bacterial and fungal communities, where changes in the abundance of dominant microorganisms, such as Ascomycota , and Mortierellomycota , etc., were closely related to soil chemistry and enzyme activity. Moreover, both significantly reduced the abundance of the pathogenic fungus Ilyonectria . Conclusions Our study clarified the effects of Allium fistulosum and Brassica napus on the microbial community and physicochemical properties of ginseng cultivated soil and provides a basis for the sustainable application of ginseng cultivation soil and the development of ginseng crop rotation systems.
 
Core Maximum likelihood phylogeny of the Methicillin-Resistant Staphylococci (MRS) isolates (2018). The phylogenetic tree was constructed using CSI-Phylogeny based on core genome SNPs extracted from alignment to reference strain LS483305.1 and visualised using Interactive Tree of Life (iTOL). SS: Staphylococcus sciuri, SE: Staphylococcus epidermidis SH: Staphylococcus haemolyticus
Location of selected livestock farms in the Northern Region of Ghana (2018)
Background The emergence of antimicrobial resistant bacteria in food producing animals is of growing concern to food safety and health. Staphylococci are common inhabitants of skin and mucous membranes in humans and animals. Infections involving antibiotic resistant staphylococci are associated with increased morbidity and mortality, with notable economic consequences. Livestock farms may enable cross-species transfer of antibiotic resistant staphylococci. The aim of the study was to investigate antimicrobial resistance patterns of staphylococci isolated from livestock and farm attendants in Northern Ghana using phenotypic and genotypic methods. Antimicrobial susceptibility testing was performed on staphylococci recovered from livestock and farm attendants and isolates resistant to cefoxitin were investigated using whole genome sequencing. Results One hundred and fifty-two staphylococci comprising S. sciuri (80%; n = 121), S. simulans (5%; n = 8), S. epidermidis (4%; n = 6), S. chromogens (3%; n = 4), S. aureus (2%; n = 3), S. haemolyticus (1%; n = 2), S. xylosus (1%; n = 2), S. cohnii (1%; n = 2), S. condimenti (1%; n = 2), S. hominis (1%; n = 1) and S. arlettae (1%; n = 1) were identified. The isolates showed resistance to penicillin (89%; n = 135), clindamycin (67%; n = 102), cefoxitin (19%; n = 29), tetracycline (15%; n = 22) and erythromycin (11%; n = 16) but showed high susceptibility to gentamicin (96%; n = 146), sulphamethoxazole/trimethoprim (98%; n = 149) and rifampicin (99%; n = 151). All staphylococci were susceptible to linezolid and amikacin. Carriage of multiple resistance genes was common among the staphylococcal isolates. Genome sequencing of methicillin (cefoxitin) resistant staphylococci (MRS) isolates revealed majority of S. sciuri (93%, n = 27) carrying mecA1 (which encodes for beta-lactam resistance) and the sal(A) gene, responsible for resistance to lincosamide and streptogramin. Most of the MRS isolates were recovered from livestock. Conclusion The study provides insights into the genomic content of MRS from farm attendants and livestock in Ghana and highlights the importance of using whole-genome sequencing to investigate such opportunistic pathogens. The finding of multi-drug resistant staphylococci such as S. sciuri carrying multiple resistant genes is of public health concern as they could pose a challenge for treatment of life-threatening infections that they may cause.
 
Background: The increase in drug-resistant opportunistic pathogenic bacteria, especially of antibiotic-resistant Staphylococcus epidermidis (S. epidermidis), has led to difficulties in the treatment of skin and soft tissue infections (SSTI). The major reason for bacterial resistance is the formation of bacterial biofilm. Here, we report a promising combination therapy of flavaspidic acid BB (BB) and mupirocin, which can effectively eradicate the biofilm of S. epidermidis and eliminate its drug resistance. Result: The susceptibility test showed that the combination of BB and mupirocin has good antibacterial and antibiofilm activities, and the fractional inhibitory concentration index (FICI) of BB combined with mupirocin was 0.51 ± 0.00 ~ 0.75 ± 0.05, showing synergistic effect. Moreover, the time-kill curve assay results indicated that the combination of drugs can effectively inhibit the planktonic S. epidermidis. After drugs treatment, the drug-combination showed significantly inhibitory effects on the metabolic activity and total biomass in each stage of biofilm formation. The synergistic effect is likely related to the adhesion between bacteria, which is confirmed by field emission scanning electron microscope. And the expression level of aap, sarA and agrA genes were detected by real-time quantitative PCR (qRT-PCR). Conclusion: Our study provides the experimental data for the use of BB for the clinical treatment of skin infections and further demonstrate the potential of BB as a novel biofilm inhibitor.
 
Distribution of neonatal patients with NDM-1 producing Klebdieella pneumoniae or Escherichia coli of common STs according to the quarters. Abbreviations: ST, sequence type; CPKP, carbapenemase-producing Klebdieella pneumoniae; CPEC, carbapenemase-producing Escherichia coli
Distribution of infant patients with NDM-1 producing Klebdieella pneumoniae strains of three representative STs according to months. Abbreviations: KP, Klebdieella pneumoniae; ST, sequence type
Background The aim of this study was to elucidate the epidemiological features of carbapenemase-producing Enterobacterales (CPE) in the pediatric and neonatal patients, to describe clinical characteristics of neonatal patients with CPE infections, and to assess risk factors for neonatal rectal colonization with CPE. Results A total of 439 carbapenem-resistant Enterobacterales (CRE) isolates recovered from 367 infant patients were characterised, including 397 isolates of Klebsiella pneumoniae (KP) and 42 isolates of Escherichia coli (EC). Carbapenemase gene blaNDM-1 was the most commonly detected, accounting for 86.56% ( n = 380), followed by blaKPC-2 (9.11%, 40) and blaIMP-4 (4.33%, 19). MLST analysis showed 17 different STs detected within CPKP isolates, with ST20, ST2068, ST36 and ST17 being the most frequently isolated types. Eleven STs were identified within CPEC isolates, with ST325 being the dominant types. Eight isolates of NDM-1 producing KP, belonging to ST23, were identified as having hypervirulent traits. The main infections caused by CPE were pneumonia ( n = 90) and sepsis ( n = 16). All infected patients received monotherapy, with meropenem and ciprofloxacin being the most commonly used antibiotics. All pneumonia patients were cured or improved after treatment. Of the 16 patients with sepsis, 9 were cured or improved, 3 died, and 4 abandoned treatment without any clinical improvement. The rectal prevalences of CPE in the 0–3 days old (DO), the 4–28 DO, and the 29 DO-1 year old groups were decreased from 15.31%, 27.37% and 14.29% in the first stool screening period to 11.78%, 19.59% and 4.07% in the second stool screening period, respectively. Multivariate analysis showed that cesarean section, acidosis, respiration failure, gastric lavage and enema were independent risk factors for rectal colonization in the 0–3 DO group, whereas cesarean section, cephalosporins, gastric lavage and residence in rural area were independently associated with rectal colonization in the 4–28 DO group. The implementation of a series of evidence-based control measures eventually contained the CPE transmission. Conclusions Continued vigilance, epidemiological studies, and multimodal infection prevention strategies are urgently needed due to frequent importations.
 
Phylogenetic analyses of concatenated hsp65 and rpoB gene sequences of clinical MAC isolates, using the neighbor-joining method by MEGA10 software. The bootstrap value marked on the node is used to evaluate the reliability of the branch. The evolutionary branch length value on the branch indicates the genetic variability of the evolutionary branch. Each species is marked with the same color: M. intracellulare (medium blue), M. avium (light coral), M. marseillense (fuchsia), M. colombiense (dark orange), M. yongonense (hot pink), isolate HZ347 (gold), and isolate 18-T1838 (aquamarine)
Background Recently, Mycobacterium avium complex (MAC) infections have been increasing, especially in immunocompromised and older adults. The rapid increase has triggered a global health concern due to limited therapeutic strategies and adverse effects caused by long-term medication. To provide more evidence for the treatment of MAC, we studied the in vitro inhibitory activities of 17 antimicrobial agents against clinical MAC isolates. Results A total of 111 clinical MAC isolates were enrolled in the study and they were identified as M. intracellulare , M. avium , M. marseillense , M. colombiense , M. yongonense , and two isolates could not be identified at the species level. MAC strains had relatively low (0–21.6%) resistance to clarithromycin, amikacin, bedaquiline, rifabutin, streptomycin, and clofazimine, and the resistant rates to isoniazid, rifampin, linezolid, doxycycline, and ethionamide were very high (72.1–100%). In addition, M. avium had a significantly higher resistance rate than that of M. intracellulare for ethambutol (92.3% vs 40.7%, P < 0.001), amikacin (15.4% vs 1.2%, P = 0.049), and cycloserine (69.2% vs 25.9%, P = 0.004). Conclusions Our results supported the current usage of macrolides, rifabutin, and aminoglycosides in the regimens for MAC infection, and also demonstrated the low resistance rate against new drugs, such as clofazimine, tedizolid, and bedaquiline, suggesting the possible implementation of these drugs in MAC treatment.
 
Stimulation of HaCaT cells with c-di-GMP inhibits MRSA colonization. A MRSA strain HRCH2019-1 was grown in TSB at 37 °C and 200 rpm in the presence of c-di-GMP (0, 1, 5, 25, or 125 μM) and bacterial growth was monitored by absorbance measurements (OD600) at 2-h intervals. B Cytotoxicity of c-di-GMP against HaCaT cells was determined with the CCK-8 assay. Viability of HaCaT cells after treatment for 24 h with c-di-GMP at 1, 5, 25 and 125 μM was determined relative to untreated control cells. C HaCaT cells were incubated for 12 h with c-di-GMP at 0, 1, 5, 25, or 125 μM and challenged with MRSA strain HRCH2019-1. Bacterial colonization of HaCaT cells was determined after 3 h incubation. Significant differences compared with 0 μM c-di-GMP were identified by one-way ANOVA with Dunnett’s multiple comparisons test (GraphPad Prism). ***, P < 0.001
Stimulation of HaCaT cells with c-di-GMP induces secretion of antimicrobial peptides and chemokines. HaCaT cells were incubated for 12 h with c-di-GMP at 0, 1, 5, 25, or 125 μM and challenged with MRSA strain HRCH2019-1 for 3 h. Culture medium of infected and uninfected control cells was harvested for quantification of antimicrobial peptides (AMPs) and chemokines by ELISA. A Quantification of human AMP β-defensin 1 (hBD-1). B Quantification of AMP hBD-2. C Quantification of AMP hBD-3. D Quantification of AMP LL-37. E Quantification of chemokine CXCL1. F Quantification of chemokine CXCL8. Significant differences compared with 0 μM c-di-GMP for both the MRSA-challenged and unchallenged cells and for other specified comparisons were identified by two-way ANOVA with Dunnett’s multiple comparisons test (GraphPad Prism). *, P < 0.05; **, P < 0.01; ***, P < 0.001
Stimulation of HaCaT cells with c-di-GMP induces gene expression of MHC class I and II. HaCaT cells were incubated for 12 h with c-di-GMP at 0, 1, 5, 25, or 125 μM and challenged with MRSA strain HRCH2019-1 for 3 h. Infected and uninfected control cells was harvested and total RNA was extracted with TRIzol. Relative gene expression of MHC class I and II was determined by quantitative real-time PCR following the 2.−ΔΔCt method with GAPDH expression for normalization. A Relative gene expression of MHC class I. B Relative gene expression of MHC class II. Significant differences compared with 0 μM c-di-GMP for both the MRSA-challenged and unchallenged cells were identified by two-way ANOVA with Dunnett’s multiple comparisons test (GraphPad Prism). ***, P < 0.001
Pre-treatment with c-di-GMP attenuates MRSA colonization in a murine skin wound infection model. Groups of mice were pre-treated with 200 nmol c-di-GMP or PBS and 24 h later damaged skin sections were challenged with MRSA strain HRCH2019-1. Challenged skin sections were excised for 3 consecutive days post infection for quantification of bacterial CFU counts and histology analysis after H&E staining. A Quantification of MRSA CFUs from challenged skin tissue sections. Significant differences between the control and c-di-GMP pre-treatment group were identified by two-way ANOVA with Sidak’s multiple comparisons test (GraphPad Prism). ***, P < 0.001. B Representative microscopy images of MRSA-challenged H&E-stained skin tissue sections. C Quantification of neutrophils in MRSA-challenged H&E-stained skin tissue sections. Significant differences between the control and c-di-GMP pre-treatment group were identified by two-way ANOVA with Sidak’s multiple comparisons test (GraphPad Prism). *, P < 0.05; **, P < 0.01
Background Staphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model. Results Stimulation of HaCaT cells with 125 μM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human β-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 μM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice. Conclusions These results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.
 
Top-cited authors
Scot E Dowd
  • Molecular Research LP. MR DNA
Randall Wolcott
  • Medical Director of Southwest Reginal Wound Care Center
Joel Dore
  • French National Institute for Agriculture, Food, and Environment (INRAE)
Gilles Vergnaud
  • Université Paris-Saclay
Harry Sokol
  • Hôpital Saint-Antoine (Hôpitaux Universitaires Est Parisien)