Preliminary study on the role of novel LysR family gene kp05372 in Klebsiella pneumoniae of forest musk deer.

LysR-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae, leading to severe infection. Earlier, we found a novel LysR family gene, named kp05372, in a strain of K. pneumoniae (designated GPKP) isolated from forest musk deer. To study the function of this gene in relation to the biological characteristics of GPKP, we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372; moreover, we also constructed the GPKP-Δkp05372+ complemented strain. The role of this gene was determined by comparing the following characteristics of three strains: growth curves, biofilm formation, drug resistance, stress resistance, median lethal dose (LD50), organ colonization ability, and the histopathology of GPKP. Real-time polymerase chain reaction (RT-PCR) was used to test the expression level of seven genes upstream of kp05372. There was no significant difference in the growth rates when comparing the three bacterial strains, and no significant difference was recorded at different osmotic pressures, temperatures, salt contents, or hydrogen peroxide concentrations. The GPKP-Δkp05372 mutant formed a weak biofilm, and the other two strains formed medium biofilm. The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin, cotrimoxazole, and polymyxin B was changed. The acid tolerance of the deletion strain was stronger than that of the other two strains. The LD50 values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant, respectively. The colonization ability of the GPKP-Δkp05372 mutant in the heart, liver, spleen, kidney, and intestine was the weakest. The three strains caused different histopathological changes in the liver and lungs. In the GPKP-Δkp05372 mutant, the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold, respectively, while the level of kp05378 was decreased by 42%. Overall, the deletion of kp05372 gene leads to changes in the following: drug resistance and acid tolerance; decreases in virulence, biofilm formation, and colonization ability of GPKP; and regulation of the upstream region of adjacent genes.

Effects of compound probiotics on the weight, immunity performance and fecal microbiota of forest musk deer.

Probiotics are intended to provide health benefits when consumed, generally by improving or restoring the gut flora. The health problems of forest musk deer (FMD, Moschus berezovskii), a threatened species currently under conservation, restrict the development of captive musk deer. This study was conducted with the aim of analyzing the effects of forest musk deer compound probiotics (FMDPs) on weight, immunity performance and fecal microbiota in FMD by measuring average daily weight gain (ADG) and immune-related factors and by using high-throughput 16S rRNA sequencing to investigate differences in the fecal microbiota among the control group (4 samples), treatment group A (4 samples) and treatment group B (4 samples). The results showed that the ADG of treatment groups A and B was significantly higher than that of the control group (p = 0.032, p = 0.018). The increase in IgA and IgG levels in treatment group B was significantly higher than that in the control group (p = 0.02, p = 0.011). At the phylum and genus levels, the difference in bacterial community structure was significant between treatment group B and the control group. Both the alpha diversity and beta diversity results showed significant differences in the microbiota of FMD before and after FMDP feeding. In summary, the results indicated that FMDPs could promote the growth of growing FMD, improve immunity and balance the role of intestinal microbes.

New genotypes and molecular characterization of Enterocytozoon bieneusi in pet birds in Southwestern China.

Enterocytozoon bieneusi, a unicellular enteric microsporidian parasite, can infect humans and a wide range of animals throughout the world. Although E. bieneusi has been identified in many animals, there is no information regarding the genotypes of E. bieneusi in pet birds in China. Birds are important sources of emerging infectious diseases that affect humans, and immunosuppressed individuals can be exposed to potential zoonotic agents shed by birds. The aim of the present study was to determine the prevalence and genotypic diversity of E. bieneusi in pet birds, as well as assessed its zoonotic potential. A total of 387 fecal samples were collected from Psittaciformes (n = 295), Passeriformes (n = 67), and Galliformes (n = 16) from four pet markets in Sichuan province, Southwestern China. The overall prevalence of E. bieneusi in pet birds was 25.1% based on nested polymerase chain reaction analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene (Psittaciformes, 21.7%; Passeriformes, 37.3%; Galliformes, 50.0%). Eight genotypes of E. bieneusi were identified, including five known genotypes (D, SC02, BEB6, CHB1, and MJ5) and three novel genotypes (SCB-I, SCB-II, and SCB-III). In phylogenetic analysis, genotypes D and SC02 and one novel genotype SCB-II were clustered within group 1, genotype BEB6 was classified within group 2, and the remaining genotypes (CHB1, MJ5, SCB-I, and SCB-III) clustered with group 10. To the best of our knowledge, this is the first report of E. bieneusi infection in pet birds in China. Genotypes D, SC02, and BEB6 that have been previously identified in humans, were found in pet birds in this study, suggesting that these pet birds can be a potential source of human microsporidiosis in China.

First report of Blastocystis in giant pandas, red pandas, and various bird species in Sichuan province, southwestern China.

Blastocystis is a common enteric protist that colonizes humans and a wide range of animals. Although some studies have reported incidences of Blastocystis in humans and animals in China, there is no information available on the prevalence of Blastocystis in giant pandas, red pandas, or bird species. The aims of the present study were to determine the prevalence, subtype distribution, and genetic characterizations of Blastocystis in these animals in a captive situation in southwestern China, as well as assess the zoonotic potential of Blastocystis isolates. A total of 168 fecal specimens, including 81 from giant pandas, 23 from red pandas, 38 from black swans, 11 from ruddy shelducks, and 15 from green peafowl were collected at the Chengdu Research Base of Giant Panda Breeding in Sichuan province. The overall minimum prevalence of Blastocystis was 11.3% (19/168) based on PCR amplification of the barcode region of the SSU rRNA gene. The highest prevalence of Blastocystis was observed in ruddy shelduck (18.2%) and the lowest was found in green peafowl (6.7%). The prevalence of Blastocystis in giant pandas >5.5 years of age was higher than that in younger giant pandas. Two potentially zoonotic subtypes (ST1 and ST8) were identified, and ST1 (n = 12) was found to be more prevalent than ST8 (n = 7). To the best of our knowledge, this is the first report of the prevalence and subtypes of Blastocystis in giant pandas, red pandas, and bird species in China. The findings of this study will improve our understanding of the genetic diversity and public health potential of Blastocystis.

SMART Design of a Bulk-Capped Supramolecular Segment for the Assembly into Organic Interdigital Lipid Bilayer-Like (ILB) Nanosheets.

Rational molecular design for the organic nanocrystal morphology still remains a challenge due to the structural diversity and complicated weak intermolecular interactions. In this work, a typical attractor-repulsor molecule N,N-diphenyl-4-(9-phenyl-fluoren-9-yl) phenylamine (TPA-PF) is designed to explore a general assembly strategy for 2D nanocrystals. Via an interdigital lipid bilayer-like (ILB) molecular packing mode, large-sized lamellar 2D nanosheets are obtained with a length:width:thickness ratio as ≈2500:1000:1. The d-spacing of the largest (001) plane is 1.32 nm, which equals to the thickness of a single interdigital stacking layer. The synergetic effect of the attractive supramolecular segment (TPA) and the repulsive bulky group (PF) is supposed to be the critical factor for the ILB packing that leads to the 2D structures. The attractor-repulsor molecule design is expected to be an effective strategy for the growth of 2D nanocrystals based on small organic molecules.

ISOLATION, IDENTIFICATION, AND GENOME ANALYSIS OF LUNG PATHOGENIC KLEBSIELLA PNEUMONIAE (LPKP) IN FOREST MUSK DEER.

Klebsiella pneumoniae is an important pathogen commonly associated with opportunistic infections. In this study, lung pathogenic K. pneumoniae (LPKP) was isolated and identified from suppurative pneumoniae in forest musk deer by conventional methods and by 16S ribosomal RNA sequence analysis. Median lethal dose and histopathologic analysis were used to demonstrate pathogenicity of the organism in mice. Furthermore, a draft genome of LPKP was sequenced, and its virulence genes were detected. One hundred and twenty-two virulence genes encoded determinant of capsule polysaccharide (CPS), lipopolysaccharide, fimbriae, outer membrane proteins, iron acquisition, and urease. In particular, 20 CPS-related genes were highly conserved in LPKP, K. pneumoniae U, K. pneumoniae NTUH-KP35, and K. pneumoniae KP-1. All of the strains were identified as capsular type K54. This is the first report of capsular type K54 K. pneumoniae causing suppurative pneumonia in an animal. The results of this study provided the basis for understanding the pathogenicity of LPKP and laid a foundation for the development of vaccines for the capsular type K54 K. pneumoniae disease.