Integrated analysis of transcriptome and metabolome reveals the regulatory mechanism of largemouth bass (Micropterus salmoides) in response to Nocardia seriolae infection.

Nocardia seriolae is a severe bacterial pathogen that has seriously affected the development of aquaculture industry. Largemouth bass (Micropterus salmoides) is a commercially significant freshwater fish that suffers a variety of environmental threats, including bacterial pathogens. However, the immune responses and metabolic alterations of largemouth bass to N. seriolae infection remain largely unclear. We discovered that N. seriolae caused pathological alterations in largemouth bass and shifted the transcript of immune-related and apoptotic genes in head kidney after infection. To answer the aforementioned question, a combined transcriptome and metabolome analysis was employed to explore the alterations in genes, metabolites, and metabolic pathways in largemouth bass following bacterial infection. A total of 3579 genes and 1929 metabolites are significant differentially changed in the head kidney post infection. In response to N. seriolae infection, host modifies the PI3K-Akt signaling pathway, TCA cycle, glycolysis, and amino acid metabolism. The integrated analysis of transcriptome and metabolome suggested that with the arginine metabolism pathway as the core, multiple biomarkers (arg gene, arginine) are involved in the antibacterial and immune functions of largemouth bass. Thus, we hypothesized that arginine plays a crucial role in the immune responses of largemouth bass against N. seriolae infection, and increasing arginine levels suitably is beneficial for the host against bacterial infection. Our results shed light on the regulatory mechanism of largemouth bass resistance to N. seriolae infection and contributed to the development of more effective N. seriolae resistance strategies.

Multiple Copies of Mobile Tigecycline Resistance Efflux Pump Gene Cluster tmexC2D2.2-toprJ2 Identified in Chromosome of Aeromonas spp.

The appearance and prevalence of novel plasmid-encoded tigecycline resistance efflux pump gene clusters tmexC1D1-toprJ1 and tmexC2D2-toprJ2 in Enterobacteriaceae have raised a threat to public health. Here, another tigecycline resistance gene cluster, tmexC2D2.2-toprJ2, was identified in two Aeromonas isolates recovered from fish meat and vegetables. Cloning confirmed the expression of tmexC2D2.2-toprJ2 mediated the resistance to tigecycline and decreased susceptibility to tetracyclines and cephalosporins in both Escherichia coli and Aeromonas. In an Aeromonas veronii strain, four copies of tmexC2D2.2-toprJ2 were located on the chromosome. Further analysis revealed that tmexC2D2.2-toprJ2 has been detected in the chromosomes of A. veronii, Aeromonas hydrophila, and Aeromonas caviae with one to four copies due to the insertion of a potential integrative transferable unit. The occurrence of multiple copies of chromosomal tmexC2D2.2-toprJ2 may act as a sink for this tigecycline resistance gene cluster, which requires continuous monitoring. IMPORTANCE Tigecycline is regarded as one of the few effective drugs against multidrug-resistant bacterial infection. However, mobile tigecycline resistance efflux pump gene clusters such as tmexC1D1-toprJ1 and its variants have been identified in both animal- and human-origin Enterobacteriaceae. In this study, we first found another efflux pump gene cluster, tmexC2D2.2-toprJ2, in the Aeromonas chromosome. This gene cluster could mediate tigecycline resistance and decrease susceptibility to tetracyclines and cephalosporins in the Aeromonas host strain. Meanwhile, tmexC2D2.2-toprJ2 was detected with multiple copies in Aeromonas spp. This multidrug resistance efflux pump gene cluster with multiple copy numbers might stably exist in Aeromonas and serve as a reservoir for tmexCD2-toprJ2, facilitating its persistent presence and spread.

The Gut Microbial Co-Abundance Gene Groups (CAGs) Differentially Respond to the Flavor (Yao-Wei) of Chinese Materia Medica.

The property theory is a unique principle instructing traditional Chinese doctors to prescribe proper medicines against diseases. As an essential part of it, the five-flavor theory catalogs various Chinese materia medicas (CMMs) into five flavors (sweet, bitter, sour, salty, and pungent) based on their taste and medical functions. Although CMM has been successfully applied in China for thousands of years, it is still a big challenge to interpret CMM flavor via modern biomarkers, further deepening its elusiveness. Herein, to identify the correlation between gut microbiota and CMM flavor, we selected 14 CMMs with different flavors to prepare their aqueous extracts, quantified the contained major chemical components, and then performed full-length 16S rRNA sequencing to analyze the gut microbiota of C57BL/6 mice administrated with CMM extracts. We found that flavones, alkaloids, and saponins were the richest components for sweet-, bitter-, and pungent-flavored CMMs, respectively. Medicines with merged flavors (bitter-pungent and sweet-pungent) displayed mixed profiles of components. According to gut microbial analysis, modulation of CMMs belonging to the same flavor on the taxonomic classification was inconsistent to an extent, while the functional sets of gut microbiota, co-abundance gene groups (CAGs), strongly and differentially responded to distinct flavors. Moreover, these correlations were in line with their pharmacological actions. Therefore, the gut microbial functional sets (CAGs) could act as the possible indicator to reflect CMM flavor, rather than the composition of microbial community.

Molecular characterization and functional analysis of IL-18 in snakehead (Channa argus) during Aeromonas schubertii and Nocardia seriolae infections.

As a proinflammatory cytokine of the interleukin-1 (IL-1) family, IL-18 plays important roles in host protection against bacterial, viral, and fungal infection. We cloned the open reading frame of snakehead (Channa argus) IL-18 (shIL-18) and found that it contained 609 base pairs and encoded 202 amino acid residues. The shIL-18 included a conserved IL-1-like family signature and two potential IL-1ß-converting enzyme cutting sites; one was conserved in all analyzed IL-18s, but the other was unique to shIL-18. Unlike other IL-18s, shIL-18 also contained a predicted signal peptide. In this study, shIL-18 was constitutively expressed in all tested tissues, and its expression was induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo and by lipoteichoic acid, lipopolysaccharides, and polyinosinic-polycytidylic acid in head kidney leukocytes in vitro. Moreover, recombinant shIL-18 upregulated the expression of interferon-γ, IL-1ß, and tumor necrosis factor-α1 and -α2 and promoted the proliferation of leukocytes. Taken together, these results showed that IL-18 played crucial roles in host defense against bacterial infection in fish, as it does in mammals.

Two types of TNF-α and their receptors in snakehead (Channa argus): Functions in antibacterial innate immunity.

Tumor necrosis factor-α (TNF-α) is a pluripotent mediator of pro-inflammatory and antimicrobial defense mechanisms and a regulator of lymphoid organ development. Although two types of TNF-α have been identified in several teleost species, their functions in pathogen infection remain largely unexplored, especially in pathogen clearance. Herein, we cloned and characterized two types of TNF-α, termed shTNF-α1 and shTNF-α2, and their receptors, shTNFR1 and shTNFR2, from snakehead (Channa argus). These genes were constitutively expressed in all tested tissues, and were induced by Aeromonas schubertii and Nocardia seriolae in head kidney and spleen in vivo, and by lipoteichoic acid (LTA), lipopolysaccharides (LPS), and Polyinosinic-polycytidylic acid [Poly (I:C)] in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shTNF-α1 and shTNF-α2 upregulated the expression of endogenous shTNF-α1, shTNF-α2, shTNFR1, and shTNFR2, and enhanced intracellular bactericidal activity, with shTNF-α1 having a greater effect than shTNF-α2. These findings suggest important roles of fish TNFα1, TNFα2, and their receptors in bacterial infection and pathogen clearance, and provide a new insight into their function in antibacterial innate immunity.

Bacteria-induced IL-1ß and its receptors in snakehead (Channa argus): Evidence for their involvement in antibacterial innate immunity.

As a central pro-inflammatory cytokine, interleukin-1ß (IL-1ß) plays critical roles in the inflammatory response, pathogen infection, and immunological challenges in mammals. Although fish IL-1ß has been confirmed to participate in inflammatory response to pathogen infection, few studies have been performed to characterize the antibacterial and bactericidal functions of fish IL-1ß. In this study, snakehead (Channa argus) IL-1ß (shIL-1ß) and its receptors, shIL-1R1 and shIL-1R2, were cloned and functionally characterized. ShIL-1ß contained the IL-1 family signature domain, and a potential cutting site at Asp96 that presented in all vertebrate IL-1ß sequences. ShIL-1R1 had three extracellular IG-like domains and one intracellular signal TIR domain, while shIL-1R2 had three extracellular IG-like domain but lacked the intracellular signal TIR domain. ShIL-1ß, shIL-1R1, and shIL-1R2 were constitutively expressed in all tested tissues, and their expressions could be induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo, and by LTA, LPS, and Poly (I:C) in head kidney leukocytes (HKLs) in vitro. Moreover, recombinant shIL-1ß upregulated the expression of endogenous shIL-1ß, shIL-R1, and shIL-R2 in snakehead HKLs, and enhanced intracellular bactericidal activity. Taken together, this study found that, like IL-1ß and its receptors in mammals, shIL-1ß and its receptors play crucial roles in antibacterial innate immunity. This provides new insight into the evolution of IL-1ß function in vertebrates.

Isolation, identification and character analysis of Streptococcus dysgalactiae from Megalobrama terminalis.

Pure bacterial cultures were isolated from different tissues of moribund Megalobrama terminalis from a high mortality event that occurred at a farm in Foshan, China. Two isolates (F2 and F3) were identified as Streptococcus dysgalactiae subsp. dysgalactiae based on morphological and biochemical detection as well as molecular analysis. In brain heart infusion broth, the best growth conditions of isolate F3 were 35ºC, salinity 5‰ and pH 7. Furthermore, infection with isolate F3 (1.2 × 106  CFU/fish) led to the death of M. terminalis and zebrafish (Danio rerio). However, isolate F3 had no obvious pathogenicity to tilapia (GIFT, Oreochromis niloticus). When the water temperature was 29ºC, the corresponding mortality rates for zebrafish infected by isolate F3 were higher than those at 23ºC. Culture for 24 and 72 hr with isolate F3 resulted in the same mortality rates for zebrafish. The antimicrobial susceptibility assay revealed that isolate F3 was susceptible to ampicillin, florfenicol and several other antibiotics but resistant to nalidixic acid, streptomycin, sulfamethoxazole/trimethoprim, neomycin and amikacin. To our knowledge, this is the first report that S. dysgalactiae infected the subtropical freshwater fish M. terminalis, which indicates that this bacterium is a potential threat to subtropical freshwater fish.

[Plan for Improving the Initial Ambulation of Acute Stroke Patients].

BACKGROUND & PROBLEMS: Acute stroke patients should receive a rehabilitation assessment within 24-48 hours of hospitalization. Initial ambulation is known to reduce the occurrence of complications, improve the ability to perform activities of daily living, and reduce the risk of long-term disability. PURPOSE: To raise the initial ambulation willingness of acute stroke patients and to increase the willingness of these patients to receive rehabilitation treatment as soon as possible in order to reduce the long-term physical damage of the stroke incident. RESOLUTIONS: To develop and implement standard operating procedures for the initiation of ambulation (first time leaving the hospital bed) in acute stroke patients, to use health education brochures with texts and illustrations, and to have nurses physically assist patients to initiate ambulation. RESULTS: The rate of ambulation initiation in acute stroke patients rose from 32.0% pre-intervention to 85.4% post-intervention. CONCLUSIONS: Acute stroke patients who initiate ambulation soon after experiencing a stroke may reduce their risk of acute complications, increase their ability to perform activities of daily living, and reduce the risk of long-term disability. Thus, encouraging early ambulation is extremely important to improving the prognosis of this patient population.

Analysis of antimicrobial resistance genes in Aeromonas spp. isolated from cultured freshwater animals in China.

The development of resistance to antimicrobials used in aquatic animals is an increasing concern for aquaculture and public health. To monitor the occurrence of antimicrobial resistance and resistance genes in Aeromonas, a total of 106 isolates were collected from cultured freshwater animals in China from 1995 to 2012. Antimicrobial susceptibilities were determined by the disk diffusion method. The highest resistance percentage occurred with ampicillin, rifampin, streptomycin, and nalidixic acid. Most strains were sensitive to fluoroquinolones, doxycycline, cefotaxime, chloramphenicol, and amikacin. The isolates from turtle samples had the highest levels of resistance to 11 of the 12 tested antimicrobials when compared with those from fish or shrimp. Polymerase chain reaction and DNA sequence results showed that all trimethoprim/sulfamethoxazole-resistant strains contained sul1, and 37.0% were positive for tetA in tetracycline-resistant strains. ant(3″)-Ia was identified in 13 (24.5%) streptomycin-resistant strains. Plasmid-borne quinolone resistance genes were detected in five Aeromonas hydrophila (4.7%), two of which carried qnrS2, while the other three strains harbored aac(6')-Ib-cr. Two cefotaxime-resistant A. hydrophila were positive for bla(TEM-1) and bla(CTX-M-3). To our knowledge, this is the first report characterizing antimicrobial resistance in Aeromonas isolated from cultured freshwater animals in China, and providing resistance information of pathogen in Chinese aquaculture.

Prevalence and characteristics of rmtB and qepA in Escherichia coli isolated from diseased animals in China.

16S rRNA methylase and QepA, a fluoroquinolone efflux pump, are new mechanisms of resistance against aminoglycosides and fluoroquinolone, respectively. One of 16S rRNA methylase genes, rmtB, was found to be associated with qepA, were both located on the same transposable element. In this study, we intended to determine the current prevalence and characteristics of the 16S rRNA methylase genes and qepA, and to study the association between rmtB and qepA. A total of 892 Escherichia coli isolates were collected from various diseased food-producing animals in China from 2004 to 2008 and screened by PCR for 16S rRNA methylase genes and qepA. About 12.6% (112/892) and 0.1% (1/892) of isolates that were highly resistant to amikacin were positive for rmtB and armA, respectively. The remaining five 16S rRNA methlyase genes were not detected. Thirty-six (4.0%) strains carried qepA. About 32.1% of rmtB-positive strains harbored qepA, which was not detected in rmtB-negative strains. Most strains were clonally unrelated, while identical PFGE profiles of rmtB-positive isolates were found in the same farm indicating clonal transmission. Conjugation experiments showed that rmtB was transferred to the recipients, and qepA also cotransferred with rmtB in some cases. The spread of E. coli of food animal origin harboring both rmtB and qepA suggests that surveillance for antimicrobial resistance of animal origin as well as the study of the mechanisms of resistance should be undertaken.

Antimicrobial resistance in Escherichia coli isolates from food animals, animal food products and companion animals in China.

One thousand and thirty Escherichia coli isolates from food animals, animals-derived foods, and companion animals between 2007 and 2008 in Southern China were used to investigate their antimicrobial susceptibility to 14 different antimicrobials by the standard agar dilution method. More than 70% of isolates showed resistance to tetracycline, trimethoprim-sulphamethoxazole, nalidixic acid, and ampicillin. In general, resistance was less frequent in companion animal isolates vs food animals isolates, but cephalosporin and amikacin resistance was more frequent in companion animal isolates, 42.6% to 56.2% vs 14.1% to 24.3%, and 28.5% vs 18.8%, respectively, which was most likely due to the common use of these antimicrobials as treatment in pet animals. Fluoroquinolones resistance was high in all animal isolates (>50%). Food products showed lowest resistance among isolates from these three resources. PFGE analysis indicated that a majority of multidrug-resistant E. coli isolates showed unique, unrelated PFGE profiles and were unlikely to be the spread of a specific clone. This study provides useful information about the prevalence of antimicrobial resistance in E. coli isolated from animals and food products in China and provided evidence of the linkage of the use of antimicrobials in animals and its selection of antimicrobial resistance in bacterial isolates. The data from this study further warns the prudent use of antimicrobials in food and pet animals to reduce the risks of transmission of antimicrobial resistance zoonotic pathogen to humans.