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Lytic bacteriophages (phages) are promising clinically viable therapeutic options against carbapenem-resistant Klebsiella pneumoniae (CRKP). In China, the predominant strains are those assigned to sequence type 11 and capsular type 64 (ST11-KL64). The emergence of phage resistance is a major bottleneck hindering effective phage therapy, requiring more new phages to provide the flexibility for creating different phage cocktails. However, the majority of phages against ST11-KL64 CRKP belong to the genus Przondovirus of the family Autographiviridae, which limits the options for constructing cocktails. We recovered a novel lytic phage of the genus Taipeivirus within the family Ackermannviridae against ST11-KL64 CRKP from a river in China. We phenotypically characterized this phage and obtained its genome sequence for analysis. This phage can inhibit the growth of ST11-KL64 CRKP for 6.5 h at a 0.1 multiplicity of infection and exhibits a narrow host range, being unable to attack CRKP strains of the other 30 capsular types. This phage carries no genes encoding antimicrobial resistance, virulence, or lysogeny. It is stable across a wide range of temperatures and pH values, making it suitable for phage therapy. Unlike other Taipeivirus phages, P01 has two tail spike proteins and a unique tail fiber protein. The distinct tail composition of this phage contributes to its activity against ST11-KL64 CRKP and its narrow host range. Taken together, we recovered a phage of a novel viral species with the potential for therapy, which expands the phage biobank against CRKP.

BackgroundIntestinal microbiota have been demonstrated to be involved in the development of NAFLD, while the relationship between the severity of NAFLD and intestinal microbiota is still not fully elucidated. Sheng-Jiang Powder (SJP) showed exact efficacy in treating SFL and great potential in regulating intestinal microbiota, but the effects need to be further addressed in NASH and liver fibrosis.ObjectivesTo investigate the differences in intestinal microbiota of NAFLD with different severity and the effect of SJP on liver damage and intestinal microbiota.DesignNAFLD mice models with different severity were induced by high-fat diet (HFD) or choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) feeding and then treated with SJP/normal saline.MethodsBiochemical blood tests, H&E/Masson/Oil Red O/IHC staining, Western blot, and 16SrDNA sequencing were performed to explore intestinal microbiota alteration in different NAFLD models and the effect of SJP on liver damage and intestinal microbiota.ResultsIntestinal microbiota alteration was detected in all NAFLD mice. SJP induced increased expression of Pparγ and alleviated liver lipid deposition in all NAFLD mice. Microbiome analysis revealed obvious changes in intestinal microbiota composition, while SJP significantly elevated the relative abundance of Roseburia and Akkermansia, which were demonstrated to be beneficial for improving inflammation and intestinal barrier function.ConclusionOur results demonstrated that SJP was effective in improving lipid metabolism in NAFLD mice, especially in mice with SFL. The potential mechanism may be associated with the regulation of intestinal microbiota.

IntroductionThe modernized lifestyle has been paralleled by an epidemic of inflammatory bowel disease (IBD). Excessive consumption of cold beverages is especially common among the modern humans. However, whether cold stress contributes directly to the gut barrier and gut–brain axis is not clear.MethodsWe conducted a cold stress model induced by cold water. The mice were treated with 14 consecutive days of intragastric cold or common water administration. We observed changes in gut transit and gut barrier in the colon. We also employed RNA sequencing-based transcriptomic analysis to identify the genes potentially driving gut injury, and simultaneously examined the gut microbiota and metabolites in the feces.ResultsWe found that cold stress disturbed the intestinal function and increased gut permeability. A set of core genes related to immune responses were consistently overexpressed in the cold stress group. Additionally, cold stress induced decreased bacterial diversity, ecological network, and increased pathogens mainly belonging to Proteobacteria. The dopamine signaling pathway-related metabolites were largely reduced in the cold stress group.ConclusionThis study revealed that cold stress could trigger an IBD-like phenotype in mice, implying that cold stress is a possible risk factor for IBD development.

IntroductionThe essence of enterotypes is to stratify the entire human gut microbiota, and dysregulation of gut microbiota is closely related to the development of colorectal adenoma. Enterotypes may therefore be a useful target for the prevention of colorectal adenoma. However, the relationship between gut microbiota and colorectal adenoma has not been fully elucidated. In this study, we aimed to analyze the differences in gut microbiome composition between adenoma and control populations.MethodsWe recruited 31 patients with colorectal adenoma and 71 non-adenoma controls. Patient demographics, risk factors, fecal samples from each subject were collected and metagenomic sequencing was performed. LEfSe analysis was used to reveal differences in intestinal microbiome composition. Multiple logistic regression analysis was used to determine the association between enterotypes and colorectal adenoma.ResultsThe results showed that Prevotella enterotype (enterotype 4) is only present in adenoma group. Logistic regression analysis showed that Prevotella enterotype was an independent risk factor for colorectal adenoma.DiscussionThe Prevotella enterotype may increase the occurrence of colorectal adenoma through inflammatory association and interference with glucose and lipid metabolism in human body. In conclusion, the differences we observed between different enterotypes add a new potential factor to the development of colorectal adenoma.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a major cause of neonatal infections with high morbidity and mortality, and clindamycin is the main antibiotic used to treat GBS infections in patients allergic to penicillin. We aimed to analyse the antibiotic sensitivity, sequence types, serotypes, virulence factors, and antibiotic resistance mechanisms of clinically isolated clindamycin-resistant S. agalactiae and provide basic data for the treatment, prevention, and control of clinical infection of S. agalactiae. A total of 110 strains of clindamycin-resistant S. agalactiae were collected from two tertiary hospitals in Hebei, China. We performed antibiotic sensitivity tests for 11 antibiotics on these strains and whole-genome sequencing analysis. All the strains were susceptible to penicillin, ampicillin, linezolid, vancomycin, tigecycline, and quinupristin–dalfopristin. Resistance to erythromycin, levofloxacin, tetracycline, and chloramphenicol were also observed. Genome sequence analysis revealed that all strains belonged to 12 sequence types (STs) related to six cloning complexes (CCs), namely CC10, CC19, CC23, CC651, CC1, and CC17. Five serotypes were identified, including IA, IB, II, III, and V. The most prominent resistance genes were mreA (100%) and ermB (81.8%). Furthermore, cfb, cylE, pavA and the gene cluster related to the pili were 100% present in all strains, followed by lmb (95.5%) and srr1 (67.2%). This study found that clindamycin-resistant S. agalactiae showed polymorphisms in molecular types and serotypes. Furthermore, multiple virulence factor genes have been identified in their genomes.

Short-chain fatty acids (SCFAs) are metabolites of gut microbes that can modulate the host inflammatory response, and contribute to health and homeostasis. Since the introduction of the gut-skin axis concept, the link between SCFAs and inflammatory skin diseases has attracted considerable attention. In this review, we have summarized the literature on the role of SCFAs in skin inflammation, and the correlation between SCFAs and inflammatory skin diseases, especially atopic dermatitis, urticaria, and psoriasis. Studies show that SCFAs are signaling factors in the gut-skin axis and can alleviate skin inflammation. The information presented in this review provides new insights into the molecular mechanisms driving gut-skin axis regulation, along with possible pathways that can be targeted for the treatment and prevention of inflammatory skin diseases.

Microbiome and their metabolites are increasingly being recognized for their role in colorectal cancer (CRC) carcinogenesis. Towards revealing new CRC biomarkers, we compared 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC–MS) metabolite analyses in 10 CRC (TCRC) and normal paired tissues (THC) along with 10 matched fecal samples (FCRC) and 10 healthy controls (FHC). The highest microbial phyla abundance from THC and TCRC were Firmicutes, while the dominant phyla from FHC and FCRC were Bacteroidetes, with 72 different microbial genera identified among four groups. No changes in Chao1 indices were detected between tissues or between fecal samples whereas non-metric multidimensional scaling (NMDS) analysis showed distinctive clusters among fecal samples but not tissues. LEfSe analyses indicated Caulobacterales and Brevundimonas were higher in THC than in TCRC, while Burkholderialese, Sutterellaceaed, Tannerellaceaea, and Bacteroidaceae were higher in FHC than in FCRC. Microbial association networks indicated some genera had substantially different correlations. Tissue and fecal analyses indicated lipids and lipid-like molecules were the most abundant metabolites detected in fecal samples. Moreover, partial least squares discriminant analysis (PLS-DA) based on metabolic profiles showed distinct clusters for CRC and normal samples with a total of 102 differential metabolites between THC and TCRC groups and 700 metabolites different between FHC and FCRC groups. However, only Myristic acid was detected amongst all four groups. Highly significant positive correlations were recorded between genus-level microbiome and metabolomics data in tissue and feces. And several metabolites were associated with paired microbes, suggesting a strong microbiota-metabolome coupling, indicating also that part of the CRC metabolomic signature was attributable to microbes. Suggesting utility as potential biomarkers, most such microbiome and metabolites showed directionally consistent changes in CRC patients. Nevertheless, further studies are needed to increase sample sizes towards verifying these findings.

Fungi are considered to be one of the wealthiest sources of bio-metabolites that can be employed for yielding novel biomedical agents. Alternaria, including parasitic, saprophytic, and endophytic species, is a kind of dark fungi that can produce a broad array of secondary metabolites (SMs) widely distributed in many ecosystems. These are categorized into polyketides, nitrogen-containing compounds, quinones, terpenes, and others based on the unique structural features of the metabolites. New natural products derived from Alternaria exhibit excellent bioactivities characterized by antibacterial, antitumor, antioxidative, phytotoxic, and enzyme inhibitory properties. Thus, the bio-metabolites of Alternaria species are significantly meaningful for pharmaceutical, industrial, biotechnological, and medicinal applications. To update the catalog of secondary metabolites synthesized by Alternaria fungi, 216 newly described metabolites isolated from Alternaria fungi were summarized with their diverse chemical structures, pharmacological activity, and possible biosynthetic pathway. In addition, possible insights, avenues, and challenges for future research and development of Alternaria are discussed.

ObjectivesTo characterize the healthy ocular surface microbiota at the species level, including cultured and uncultured taxa.MethodsWe integrated the metataxonomic method with culturomics and genome sequencing analysis of selected isolated strains to better illustrate the taxonomic structure of the ocular surface microbiota. The metataxonomics used the full-length 16S rRNA gene sequences and the operational phylogenetic unit strategy, which can precisely identify the cultured and uncultured or potentially new taxa to species level based on the phylogenetic tree constructed.ResultsWe detected 1,731 operational phylogenetic units (OPUs) in 196 healthy eyes from 128 people, affiliated to 796 cultured species, 784 potentially new species, and 151 potentially new higher taxa. The microbiota for each eye had 49.17 ± 35.66 OPUs. Of the 796 cultured species, 170 (21.36%) had previously caused clinical infections. Based on where they were initially isolated, the ocular surface microbiota mainly came from human body sites (34.55%), the environment (36.93%), plants (9.05%), animals (4.90%), and others; 428 strains were isolated from 20 eyes, affiliated to 42 species, and had come from the environment (33.33%) and the skin (16.67%). Of these, 47.62% had previously caused clinical infections. Genome analysis of 73 isolators revealed that 68.5% of them carried antibiotic resistance genes. The most frequently isolated genera, namely Staphylococcus, Streptococcus, and Moraxella, had an average of 5.30, four, and three resistance genes per strain, respectively.DiscussionThe study found that the ocular surface microbiota mainly came from the environment, plants, animals, food, and human body sites such as the skin, oral cavity, upper respiratory tract, etc. No core member of ocular surface microbiota was detected at the species level. The human eyes were invaded and colonized by bacteria from the exposed environment, some of which were capable of causing infections in humans and carried antibiotic resistance genes. Preventive measures should be developed to protect our eyes from danger.

Avian coccidiosis is an important intestinal protozoan disease that has caused major economic losses to the poultry industry. Clostridium butyricum can not only maintain the stability of the intestinal barrier, but can also improve the production performance of broiler chickens. We studied the effects of feeding C. butyricum alone, administration of coccidiosis vaccine alone, and the combined administration of C. butyricum and coccidiosis vaccine on body weight gain, feed consumption, and feed conversion ratio of broilers. Meanwhile, intestinal contents of 8- and 15-day-old broilers were collected, and their intestinal microbiome was characterized by high-throughput sequencing of the V3–V4 region of 16S rDNA. We analyzed the oocysts per gram values and lesion scores in the C. butyricum alone group, in a group challenged with the coccidiosis-causing parasite, Eimeria, and in groups simultaneously challenged Eimeria and pretreated with C. butyricum, the coccidiosis vaccine, or combined C. butyricum and coccidiosis vaccine. Intestinal tissue samples were collected from 32-day-old broilers for microbiome analysis. Our results showed that combination of C. butyricum with coccidiosis vaccine significantly improved the performance of broiler chickens and also significantly reduced the oocysts per gram value and intestinal lesions caused by Eimeria sp. infection. Furthermore, C. butyricum and coccidiosis vaccine administered alone or in combination significantly increased the relative abundance of the immune biomarker genus Barnesiella. The significant increase in the abundance of the Clostridia_UCG.014, Eubacterium coprostanoligenes group and Bacteroides was a key factor in controlling Eimeria sp. infection.

Revealing community assembly and their impacts on ecosystem service is a core issue in microbial ecology. However, what ecological factors play dominant roles in phyllosphere fungal community assembly and how they link to crop quality are largely unknown. Here, we applied internal transcriptional spacer high-throughput sequencing to investigate foliar fungal community assembly across three cultivars of a Solanaceae crop (tobacco) and two planting regions with different climatic conditions. Network analyses were used to reveal the pattern in foliar fungal co-occurrence, and phylogenetic null model analysis was used to elucidate the ecological assembly of foliar fungal communities. We found that the sensory quality of crop leaves and the composition of foliar fungal community varied significantly across planting regions and cultivars. In Guangcun (GC), a region with relatively high humidity and low precipitation, there was a higher diversity and more unique fungal species than the region of Wuzhishan (WZS). Further, we found that the association network of foliar fungal communities in GC was more complex than that in WZS, and the network properties were closely related to the sensory quality of crop. Finally, the results of the phylogenetic analyses show that the stochastic processes played important roles in the foliar fungal community assembly, and their relative importance was significantly correlated with the sensory quality of crop leaves, which implies that ecological assembly processes could affect crop quality. Taken together, our results highlight that climatic conditions, and plant cultivars play key roles in the assembly of foliar fungal communities and crop quality, which enhances our understanding of the connections between the phyllosphere microbiome and ecosystem services, especially in agricultural production.

Long-term conventional shallow tillage reduced soil quality and limited the agriculture development. Intermittent deep tillage could effectively promote agricultural production, through optimizing soil structure, underground ecology system, and soil fertility. However, the microecological mechanism of intermittent deep tillage promoting agriculture production has never been reported, and the effect of tillage depth on crop growth has not been explored in detail. In this study, three levels of intermittent deep tillage (30, 40, and 50 cm) treatments were conducted in an experimental field site with over 10 years of conventional shallow tillage (20 cm). Our results indicated that intermittent deep tillage practices helped to improve plant physiological growth status, chlorophyll a, and resistance to diseases, and the crop yield and value of output were increased with the deeper tillage practices. Crop yield (18.59%) and value of output (37.03%) were highest in IDT-50. There were three mechanisms of intermittent deep tillage practices that improved crop growth: (1) Intermittent deep tillage practices increased soil nutrients and root system architecture traits, which improved the fertility and nutrient uptake of crop through root system. (2) Changing rhizosphere environments, especially for root length, root tips, pH, and available potassium contributed to dissimilarity of bacterial communities and enriched plant growth-promoting species. (3) Functions associated with stress tolerance, including signal transduction and biosynthesis of other secondary metabolites were increased significantly in intermittent deep tillage treatments. Moreover, IDT-30 only increased soil characters and root system architecture traits compared with CK, but deeper tillage could also change rhizosphere bacterial communities and functional profiles. Plant height and stem girth in IDT-40 and IDT-50 were higher compared with IDT-30, and infection rates of black shank and black root rot in IDT-50 were even lower in IDT-40. The study provided a comprehensive explanation into the effects of intermittent deep tillage in plant production and suggested an optimal depth.

Herein, we describe a novel bunyavirus, oriental wenrivirus 1 (OWV1), discovered in moribund oriental shrimp (Penaeus chinensis) collected from a farm in China in 2016. Like most bunyaviruses, OWV1 particles were enveloped, spherical- to ovoid-shaped, and 80–115 nm in diameter. However, its genome was found to comprise four segments of (-)ssRNA. These included an L RNA segment (6,317 nt) encoding an RNA-directed RNA polymerase (RdRp) of 2,052 aa, an M RNA segment (2,978 nt) encoding a glycoprotein precursor (GPC) of 922 aa, an S1 RNA segment (1,164 nt) encoding a nucleocapsid (N) protein of 243 aa, and an S2 RNA segment (1,382 nt) encoding a putative non-structural (NSs2) protein of 401 aa. All the four OWV1 RNA segments have complementary terminal decanucleotides (5′-ACACAAAGAC and 3′-UGUGUUUCUG) identical to the genomic RNA segments of uukuviruses and similar to those of phleboviruses and tenuiviruses in the Phenuiviridae. Phylogenetic analyses revealed that the RdRp, GPC, and N proteins of OWV1 were closely related to Wēnzhōu shrimp virus 1 (WzSV-1) and Mourilyan virus (MoV) that infect black tiger shrimp (P. monodon). Phylogenetic analyses also suggested that OWV1 could be classified into a second, yet to be established, species of the Wenrivirus genus in the Phenuiviridae. These wenriviruses also clustered with Wenling crustacean virus 7 from shrimps and bunya-like brown spot virus from white-clawed crayfish. Of note there were no homologs of the NSs2 of OWV1 and MoV/WzSV-1 in GenBank, and whether other crustacean phenuiviruses also possess a similar S2 RNA segment warrants further investigation. In addition, we established a TaqMan probe-based reverse-transcription quantitative PCR method for detection of OWV1, and it was detected as 1.17 × 102—1.90 × 107 copies/ng-RNA in gills of 23 out of 32 P. chinensis samples without an obvious gross sign. However, the discovery of OWV1 highlights the expanding genomic diversity of bunyaviruses.

BackgroundThere have been reports of increasing azole resistance in Candida tropicalis, especially in the Asia-Pacific region. Here we report on the epidemiology and antifungal susceptibility of C. tropicalis causing invasive candidiasis in China, from a 9-year surveillance study.MethodsFrom August 2009 to July 2018, C. tropicalis isolates (n = 3702) were collected from 87 hospitals across China. Species identification was carried out by mass spectrometry or rDNA sequencing. Antifungal susceptibility was determined by Clinical and Laboratory Standards Institute disk diffusion (CHIF-NET10–14, n = 1510) or Sensititre YeastOne (CHIF-NET15–18, n = 2192) methods.ResultsOverall, 22.2% (823/3702) of the isolates were resistant to fluconazole, with 90.4% (744/823) being cross-resistant to voriconazole. In addition, 16.9 (370/2192) and 71.7% (1572/2192) of the isolates were of non-wild-type phenotype to itraconazole and posaconazole, respectively. Over the 9 years of surveillance, the fluconazole resistance rate continued to increase, rising from 5.7 (7/122) to 31.8% (236/741), while that for voriconazole was almost the same, rising from 5.7 (7/122) to 29.1% (216/741), with no significant statistical differences across the geographic regions. However, significant difference in fluconazole resistance rate was noted between isolates cultured from blood (27.2%, 489/1799) and those from non-blood (17.6%, 334/1903) specimens (P-value < 0.05), and amongst isolates collected from medical wards (28.1%, 312/1110) versus intensive care units (19.6%, 214/1092) and surgical wards (17.9%, 194/1086) (Bonferroni adjusted P-value < 0.05). Although echinocandin resistance remained low (0.8%, 18/2192) during the surveillance period, it was observed in most administrative regions, and one-third (6/18) of these isolates were simultaneously resistant to fluconazole.ConclusionThe continual decrease in the rate of azole susceptibility among C. tropicalis strains has become a nationwide challenge in China, and the emergence of multi-drug resistance could pose further threats. These phenomena call for effective efforts in future interventions.

For decades, more and more long non-coding RNAs (lncRNAs) have been confirmed to play important functions in key biological processes of different organisms. At present, most identified lncRNAs and those with known functional roles are from mammalian systems. However, lncRNAs have also been found in primitive eukaryotic fungi, and they have different functions in fungal development, metabolism, and pathogenicity. In this review, we highlight some recent researches on lncRNAs in the primitive eukaryotic fungi, particularly focusing on the identification of lncRNAs and their regulatory roles in diverse biological processes.

The number of antibiotics that are appropriate for Helicobacter pylori eradication in children is limited. Profiling regional or population-specific antibiotic resistance is essential in guiding the H. pylori eradication treatment in children. The aim of this study was to evaluate the antibiotic resistance in H. pylori strains isolated from children and adolescents in Southwest China. Gastric biopsies from 157 pediatric patients with or without previous H. pylori eradication treatment were collected for H. pylori culture. Susceptibility to amoxicillin (AML), clarithromycin (CLR), metronidazole (MTZ), levofloxacin (LEV), tetracycline (TET), furazolidone (FZD), and rifampicin (RIF) was determined by E-test or a disk diffusion assay. A total of 87 patients from three ethnic groups (Han/Tibetan/Yi) were H. pylori culture positive (55.4%). The overall resistance rates were 55.2% for CLR, 71.3% for MTZ, 60.9% for RIF, and 18.4% for LEV. No isolate was found to be resistant to AML, TET, and FZD. Among the 53 treatment-naïve pediatric patients, primary resistance rates to clarithromycin, metronidazole, levofloxacin, and rifampicin were 45.3, 73.6, 15.1, and 60.4%, respectively. Among the 34 treatment-experienced patients, secondary resistance rates to clarithromycin, metronidazole, levofloxacin, and rifampicin were 70.6, 67.6, 23.5, and 61.8%, respectively. Isolates exhibiting simultaneous resistance to clarithromycin and metronidazole were 28.3 and 52.9% among the treatment-naïve and treatment-experienced patients, respectively. In conclusion, among pediatric patients in Southwest China, resistance rates were high for clarithromycin, metronidazole, levofloxacin, and rifampicin, whereas nil resistance was found to amoxicillin, tetracycline, and furazolidone. Our data suggest that the standard clarithromycin-based triple therapy should be abandoned as empiric therapy, whereas the bismuth quadruple therapy (bismuth/PPI/amoxicillin/tetracycline) would be suitable as first-line empiric treatment regimen for this pediatric population. Tetracycline and furazolidone may be considered for treating refractory H. pylori infections in adolescent patients.

Escherichia coli sequence type 131 (ST131) is well known for its multidrug resistance profile. Carbapenems have been considered the treatment of choice for E. coli ST131 infections, and resistance to carbapenems is emerging due to the acquisition of carbapenemase-encoding genes. In this study, 45 carbapenem-resistant E. coli strains were collected in a hospital. The resistance mechanisms, plasmid profiles, and genetic relatedness of these strains were determined. Phylogenetic relationships between these strains were assessed by molecular profiling and aligned with patient clinical details. The genetic context of blaKPC–2 was analyzed to trace the potential dissemination of blaKPC–2. The 45 carbapenem-resistant E. coli ST131 strains were closely related. Initially prevalent only in a single ward, ST131 subsequently spread to other ward, resulting in a respiratory infection outbreak of carbapenem-resistant E. coli ST131. Eight of the 30 patients died within 28 days of the first isolation of E. coli ST131. The blaKPC–2-positive plasmid profiles suggest that the carbapenem resistance was due to the acquisition by E. coli ST131 of transmissible plasmids pE0272_KPC and pE0171_KPC carrying blaKPC–2. Additionally, diverse multidrug resistance elements were transferred and rearranged between these plasmids mediated by IS26. Our research indicates that clinical attention should be paid to the importance of E. coli ST131 in respiratory infections and the spread of blaKPC-carrying E. coli ST131.

On-site detection demands are quickly increasing to control foodborne pathogenic bacteria along with the long food supply chains. Combining the isothermal recombinase polymerase amplification (RPA) with lateral flow strips (LFSs) is a promising molecular detection approach for the short reaction time, low isothermal condition, and simple and “instrument-free” procedure. However, the method comes with a non-negligible intrinsic risk of the primer-dependent artifacts. In this study, with an important foodborne pathogenic bacterium Salmonella enterica serotype Typhimurium (S. Typhimurium) as the model, system measures including the careful selection of primers targeting unique virulence genes, use of a probe in the RPA reaction, introducing base substitutions with specific guidelines in the primer and probe sequences, and analyzing and screening the primer–probe complex formation were taken to eliminate the primer-dependent artifacts. The measures were strictly tested for the efficacy, and the standardized method was able to specifically detect S. typhimurium within 30 min at 42°C without any interference of probe–primer signals. The established RPA-LFS method shared high sensitivity with the detection limit of 1 CFU/μl of unpurified culture. Our study provided practical measures for the prevention of false positive signals from primer–dimers or primer–probe complexes when using the RPA–LFS method in pathogen detections, and also established a readily applicable method for S. Typhimurium detection.

Hand, foot, and mouth disease (HFMD) is a common viral illness affecting infants and children that is usually caused by Coxsackievirus A16 (CVA-16). To diagnose HFMD, we developed a method for rapid detection of CVA-16 based on reverse transcription-polymerase spiral reaction (RT-PSR). We used two pairs of primers that specifically recognize the conserved sequences of VP1 coding region of CVA-16, and template RNA was reverse transcribed and amplified in a single tube under isothermal conditions, total reaction time could be reduced to less than 40 min. The detection limit of this method was between 2.4 × 102 and 2.4 × 101 copies/μl with excellent specificity. To test the clinical applicability of the method, 40 clinical stool samples were analyzed using RT-PSR and quantitative reverse transcription-polymerase chain reaction, and comparison showed that the coincidence rate was 100%. Compared with other similar detection methods, RT-PSR requires less time, simpler operation, and lower cost. These results prove that our novel, simple, and reliable isothermal nucleic acid testing assay has potential application for clinical detection of CVA-16.

In fungi, peptidases play a crucial role in adaptability. At present, the roles of peptidases in ultraviolet (UV) and thermal tolerance are still unclear. In this study, a C69-family cysteine dipeptidase of the entomopathogenic fungus Metarhizium acridum, MaPepDA, was expressed in Escherichia coli. The purified enzyme had a molecular mass of 56-kDa, and displayed a high activity to dipeptide substrate with an optimal Ala-Gln hydrolytic activity at about pH 6.0 and 55°C. It was demonstrated that MaPepDA is an intracellular dipeptidase localized in the cytosol, and that it is expressed during the whole fungal growth. Disruption of the MaPepDA gene increased conidial germination, growth rate, and significantly improved the tolerance to UV-B and heat stress in M. acridum. However, virulence and conidia production was largely unaffected in the ΔMaPepDA mutant. Digital gene expression data revealed that the ΔMaPepDA mutant had a higher UV-B and heat-shock tolerance compared to wild type by regulating transcription of sets of genes involved in cell surface component, cell growth, DNA repair, amino acid metabolism, sugar metabolism and some important signaling pathways of stimulation. Our results suggested that disruption of the MaPepDA could potentially improve the performance of fungal pesticides in the field application with no adverse effect on virulence and conidiation.

Listeria monocytogenes is an important foodborne pathogenic bacterium that is explicitly threatening public health and food safety. Rapid, simple, and sensitive detection methods for this pathogen are of urgent need for the increasing on-site testing demands. Application of the isothermal recombinase polymerase amplification (RPA) and the lateral flow strip (LFS) in the detection is promising for fast speed, high sensitivity, and little dependency on equipment and trained personnel. However, the simplicity comes with an intrinsic and non-negligible risk, the false-positive signals from primer–dimers. In this study, an improved RPA–LFS system was established for detection of L. monocytogenes that eliminated false-positive signals from primer–dimers. Primer candidates were carefully selected from the entire L. monocytogenes genome sequence and rigorously screened for specific amplifications in PCR and RPA reactions. For the optimal primer pairs, probes that matched the targeted fragment sequences, although had the smallest chance to form cross-dimers with the primers, were designed and screened. The intelligent use of the probe successfully linked the positive signal to the actual amplification product. This RPA–LFS system was highly specific to L. monocytogenes and was able to detect as low as 1 colony-forming unit of the bacterium per reaction (50 μl) without DNA purification, or 100 fg of the genomic DNA/50 μl. The amplification could be conducted under the temperature between 37 and 42°C, and the whole detection finished within 25 min. Test of artificially contaminated milk gave 100% accuracy of detection without purification of the samples. Various food samples spiked with 10 colony-forming unit of L. monocytogenes per 25 g or 25 ml were successfully detected after an enrichment time period of 6 h. The RPA–LFS system established in this study is a rapid, simple, and specific detection method for L. monocytogenes that has eliminated false-positive results from primer–dimers. In addition, this study has set a good example of eliminating the false-positive risk from primer–dimers in isothermal amplification-based detection methods, which is applicable to the development of detection technologies for other pathogens.

Porcine delta coronavirus (PDCoV) is a novel emerging enterocytetropic virus causing diarrhea, vomiting, dehydration, and mortality in suckling piglets. Long non-coding RNAs (lncRNAs) are known to be important regulators during virus infection. Here, we describe a comprehensive transcriptome profile of lncRNA in PDCoV-infected swine testicular (ST) cells. In total, 1,308 annotated and 1,190 novel lncRNA candidate sequences were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these lncRNAs might be involved in numerous biological processes. Clustering analysis of differentially expressed lncRNAs showed that 454 annotated and 376 novel lncRNAs were regulated after PDCoV infection. Furthermore, we constructed a lncRNA-protein-coding gene co-expression interaction network. The KEGG analysis of the co-expressed genes showed that these differentially expressed lncRNAs were enriched in pathways related to metabolism and TNF signaling. Our study provided comprehensive information about lncRNAs that would be a useful resource for studying the pathogenesis of and designing antiviral therapy for PDCoV infection.

Coxsackievirus A4 (CVA4) is one of the most prevalent pathogens associated with hand, foot and mouth disease (HFMD), an acute febrile illness in children, and is also associated with acute localized exanthema, myocarditis, hepatitis and pancreatitis. Despite this, limited CVA4 genome sequences are currently available. Herein, complete genome sequences from CVA4 strains (n = 21), isolated from patients with HFMD in Shandong province, China between 2014 and 2016, were determined and phylogenetically characterized. Phylogenetic analysis of the VP1 gene from a larger CVA4 collection (n = 175) showed that CVA4 has evolved into four separable genotypes: A, B, C, and D; and genotype D could be further classified in to two sub-genotypes: D1 and D2. Each of the 21 newly described genomes derived from isolates that segregated with sub-genotype D2. The CVA4 genomes displayed significant intra-genotypic genetic diversity with frequent synonymous substitutions occurring at the third codon positions, particularly within the P2 region. However, VP1 was relatively stable and therefore represents a potential target for molecular diagnostics assays and also for the rational design of vaccine epitopes. The substitution rate of VP1 was estimated to be 5.12 × 10-3 substitutions/site/year, indicative of ongoing CVA4 evolution. Mutations at amino acid residue 169 in VP1 gene may be responsible for differing virulence of CVA4 strains. Bayesian skyline plot analysis showed that the population size of CVA4 has experienced several dynamic fluctuations since 1948. In summary, we describe the phylogenetic and molecular characterization of 21 complete genomes from CVA4 isolates which greatly enriches the known genomic diversity of CVA4 and underscores the need for further surveillance of CVA4 in China.

Increasing evidence has manifested that the gut bacterial microbiota of shrimps is closely related to the environmental factors, host developmental stage and health status like that of humans and animals does. These studies have provided an important guidance for improving shrimp culture benefits. In practice, aside from bacteria, eukaryotic microorganisms dominated by fungal microbiota (mycobiota), also play a key role in host growth, metabolism and homeostasis. However, little so far is known about the mycobiota in the digestive tract of shrimp. In this study, we used high-throughput sequencing of internal transcribed spacer 1 region to characterize the hepatopancreas and intestinal mycobiota of Pacific white shrimp and their connections with disease incidence and seasonal variation. The results showed that the hepatopancreas and intestinal mycobiota of Litopenaeus vannamei are dominated by the phyla Ascomycota and Basidiomycota, and the genera Alternaria, Tuber, Hortaea, Sarocladium, and Stagonospora. The fungal microbiota significantly varies under the influence of disease and seasonal variation. Sick shrimps had a higher level of potential pathogenic fungus, Candida in the intestine. Healthy shrimps had a higher abundance of the genera Didymella and Filobasidium in the gut, and Pyrenochaetopsis in the hepatopancreas. Of note, most of the fungi carried by Pacific white shrimps were pathogens to humans. This study has revealed the intestinal and hepatopancreas mycobiota of L. vannamei and the effects of diseases and seasonal variation to the mycobiota. Our study provides important guidance for Pacific white shrimp farming and sheds further insight on the fungal microbiota.

Extracellular electron transfer (EET) plays a fundamental role in microbial reduction/oxidation of minerals. Extracellular polymeric substances (EPS) surrounding the cells constitute a matrix that separates the cell’s outer membrane from insoluble minerals and environmental fluid. This study investigated the effects of EPS on EET processes during microbial reduction of hematite by the iron-reducing strain Shewanella oneidensis MR-1 (MR-1). Electrochemical characterization techniques were employed to determine the influence of EPS components on the redox ability of MR-1. Cells with removed EPS exhibited approximately 30% higher hematite reduction than regular MR-1 cells, and produced a current density of 56 μA cm-2, corresponding to 3–4 fold that of regular MR-1. The superior EET of EPS-deprived cells could be attributed to direct contact between outer membrane proteins and hematite surface, as indicated by more redox peaks being detected by cyclic voltammetry and differential pulse voltammetry. The significantly reduced current density of MR-1 cells treated with proteinase K and deoxyribonuclease suggests that the electron transfer capacity across the EPS layer depends mainly on the spatial distribution of specific proteins and electron shuttles. Exopolysaccharides in EPS tend to inhibit electron transfer, however they also favor the attachment of cells onto hematite surfaces. Consistently, the charge transfer resistance of cells lacking EPS was only 116.3 Ω, approximately 44 times lower than that of regular cells (5,139.1 Ω). These findings point to a negative influence of EPS on EET processes for microbial reduction/oxidation of minerals.

Pseudomonas putida (P. putida) MnB1 is a widely used model strain in environment science and technology for determining microbial manganese oxidation. Numerous studies have demonstrated that the growth and metabolism of P. putida MnB1 are influenced by various environmental factors. In this study, we investigated the effects of hydrogen peroxide (H2O2) and manganese (Mn2+) on proliferation, Mn2+ acquisition, anti-oxidative system, and biofilm formation of P. putida MnB1. The related orthologs of 4 genes, mco, mntABC, sod, and bifA, were amplified from P. putida GB1 and their involvement were assayed, respectively. We found that P. putida MnB1 degraded H2O2, and quickly recovered for proliferation, but its intracellular oxidative stress state was maintained, with rapid biofilm formation after H2O2 depletion. The data from mco, mntABC, sod and bifA expression levels by qRT-PCR, elucidated a sensitivity toward bifA-mediated biofilm formation, in contrary to intracellular anti-oxidative system under H2O2 exposure. Meanwhile, Mn2+ ion supply inhibited biofilm formation of P. putida MnB1. The expression pattern of these genes showed that Mn2+ ion supply likely functioned to modulate biofilm formation rather than only acting as nutrient substrate for P. putida MnB1. Furthermore, blockade of BifA activity by GTP increased the formation and development of biofilms during H2O2 exposure, while converse response to Mn2+ ion supply was evident. These distinct cellular responses to H2O2 and Mn2+ provide insights on the common mechanism by which environmental microorganisms may be protected from exogenous factors. We postulate that BifA-mediated biofilm formation but not intracellular anti-oxidative system may be a primary protective strategy adopted by P. putida MnB1. These findings will highlight the understanding of microbial adaptation mechanisms to distinct environmental stresses.

Escherichia albertii is an emerging member of the Enterobacteriaceae causing human and animal enteric infections. Antimicrobial resistance among enteropathogens has been reported to be increasing in the past years. The purpose of this study was to investigate antibiotic resistance and resistance genes in E. albertii isolated from Zigong city, Sichuan province, China. The susceptibility to 21 antimicrobial agents was determined by Kirby–Bauer disk diffusion method. The highest prevalence was tetracycline resistance with a rate of 62.7%, followed by resistance to nalidixic acid and streptomycin with a rate of 56.9 and 51.0%, respectively. All isolates were sensitive or intermediate susceptible to imipenem, meropenem, amoxicillin–clavulanic acid, and levofloxacin. Among 51 E. albertii isolates, 15 were extended-spectrum β-lactamase-producing as confirmed by the double disk test. The main β-lactamase gene groups, i.e., blaTEM, blaSHV, and blaCTX-M, were detected in17, 20, and 22 isolates, respectively. Furthermore, four colistin-resistant isolates with minimum inhibitory concentrations of 8 mg/L were identified. The colistin-resistant isolates all harbored mcr-1 and blaCTX-M-55. Genome sequencing showed that E. albertii strain SP140150 carried mcr-1 and blaCTX-M-55 in two different plasmids. This study provided significant information regarding antibiotic resistance profiles and identified the co-occurrence of β-lactamase and MCR-1 encoding genes in E. albertii isolates.

The soil microbial communities play an important role in plant health, however, the relationship between the below-ground microbiome and above-ground plant health remains unclear. To reveal such a relationship, we analyzed soil microbial communities through sequencing of 16S rRNA gene amplicons from 15 different tobacco fields with different levels of wilt disease in the central south part of China. We found that plant health was related to the soil microbial diversity as plants may benefit from the diverse microbial communities. Also, those 15 fields were grouped into ‘healthy’ and ‘infected’ samples based upon soil microbial community composition analyses such as unweighted paired-group method with arithmetic means (UPGMA) and principle component analysis, and furthermore, molecular ecological network analysis indicated that some potential plant-beneficial microbial groups, e.g., Bacillus and Actinobacteria could act as network key taxa, thus reducing the chance of plant soil-borne pathogen invasion. In addition, we propose that a more complex soil ecology network may help suppress tobacco wilt, which was also consistent with highly diversity and composition with plant-beneficial microbial groups. This study provides new insights into our understanding the relationship between the soil microbiome and plant health.

H9N2 avian influenza virus (AIV) of genotype S frequently donate internal genes to facilitate the generation of novel reassortants such as H7N9, H10N8, H5N2 and H5N6 AIVs, posing an enormous threat to both human health and poultry industry. However, the pathogenicity and transmission of reassortant H5 viruses with internal gene cassette of genotype S H9N2-origin in chickens and mice remain unknown. In this study, four H5 reassortants carrying the HA and NA genes from different clades of H5 viruses and the remaining internal genes from an H9N2 virus of the predominant genotype S were generated by reverse genetics. We found that all four H5 reassortant viruses showed attenuated virulence in both chickens and mice, thus leading to increased the mean death times compared to the corresponding parental viruses. Consistently, the polymerase activity and replication ability in mammalian and avian cells, and the cytokine responses in the lungs of chickens and mice were also decreased when compared to their respective parental viruses. Moreover, these reassortants transmitted from birds to birds by direct contact but not by an airborne route. Our data indicate that the internal genes as a whole cassette from genotype S H9N2 viruses play important roles in reducing the pathogenicity of the H5 recombinants in chickens and mice, and might contribute to the circulation in avian or mammalian hosts.

The crocodile lizard is a critically endangered reptile, and serious diseases have been found in this species in recent years, especially in captive lizards. Whether these diseases are caused by changes in the gut microbiota and the effect of captivity on disease remains to be determined. Here, we examined the relationship between the gut microbiota and diet and disease by comparing the fecal microbiota of wild lizards with those of sick and healthy lizards in captivity. The gut microbiota in wild crocodile lizards was consistently dominated by Proteobacteria (∼56.4%) and Bacteroidetes (∼19.1%). However, the abundance of Firmicutes (∼2.6%) in the intestine of the wild crocodile lizards was distinctly lower than that in other vertebrates. In addition, the wild samples from Guangdong Luokeng Shinisaurus crocodilurus National Nature Reserve also had a high abundance of Deinococcus–Thermus while the wild samples from Guangxi Daguishan Crocodile Lizard National Nature Reserve had a high abundance of Tenericutes. The gut microbial community in loach-fed crocodile lizards was significantly different from the gut microbial community in the earthworm-fed and wild lizards. In addition, significant differences in specific bacteria were detected among groups. Notably, in the gut microbiota, the captive lizards fed earthworms resulted in enrichment of Fusobacterium, and the captive lizards fed loaches had higher abundances of Elizabethkingia, Halomonas, Morganella, and Salmonella, all of which are pathogens or opportunistic pathogens in human or other animals. However, there is no sufficient evidence that the gut microbiota contributes to either disease A or disease B. These results provide a reference for the conservation of endangered crocodile lizards and the first insight into the relationship between disease and the gut microbiota in lizards.

The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa, which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa. The test times for onetube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPACas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes. [ABSTRACT FROM AUTHOR]