Pasteurella multocida strains of a novel capsular serotype and lethal to Marmota himalayana on Qinghai-Tibet plateau in China.

Pasteurella multocida is a zoonotic pathogen causing serious diseases in humans and animals. Here, we report P. multocida from wildlife on China's Qinghai-Tibet plateau with a novel capsular serotype, forming a single branch on the core-genome phylogenetic tree: four strains isolated from dead Himalayan marmot (Marmota himalayana) and one genome assembled from metagenomic sequencing of a dead Woolly hare (Lepus oiostolus). Four of the strains were identified as subspecies multocida and one was septica. The mouse model showed that the challenge strain killed mice within 24 h at an infectious dose of less than 300 bacteria. The short disease course is comparable to septicemic plague: the host has died before more severe pathological changes could take place. Though pathological changes were relatively mild, cytokine storm was obvious with a significant rise of IL-12p70, IL-6, TNF-αand IL-10 (P < 0.05). Our findings suggested P. multocida is a lethal pathogen for wildlife on Qinghai-Tibet plateau, in addition to Yersinia pestis. Individuals residing within the M. himalayana plague focus are at risk for P. multocida infection, and public health warnings are necessitated.

Metagenomics of gut microbiome for migratory seagulls in Kunming city revealed the potential public risk to human health.

BACKGROUND: Seagull as a migratory wild bird has become most popular species in southwest China since 1980s. Previously, we analyzed the gut microbiota and intestinal pathogenic bacteria configuration for this species by using 16S rRNA sequencing and culture methods. To continue in-depth research on the gut microbiome of migratory seagulls, the metagenomics, DNA virome and RNA virome were both investigated for their gut microbial communities of abundance and diversity in this study. RESULTS: The metagenomics results showed 99.72% of total species was bacteria, followed by viruses, fungi, archaea and eukaryota. In particular, Shigella sonnei, Escherichia albertii, Klebsiella pneumonia, Salmonella enterica and Shigella flexneri were the top distributed taxa at species level. PCoA, NMDS, and statistics indicated some drug resistant genes, such as adeL, evgS, tetA, PmrF, and evgA accumulated as time went by from November to January of the next year, and most of these genes were antibiotic efflux. DNA virome composition demonstrated that Caudovirales was the most abundance virus, followed by Cirlivirales, Geplafuvirales, Petitvirales and Piccovirales. Most of these phages corresponded to Enterobacteriaceae and Campylobacteriaceae bacterial hosts respectively. Caliciviridae, Coronaviridae and Picornaviridae were the top distributed RNA virome at family level of this migratory animal. Phylogenetic analysis indicated the sequences of contigs of Gammacoronavirus and Deltacoronavirus had highly similarity with some coronavirus references. CONCLUSIONS: In general, the characteristics of gut microbiome of migratory seagulls were closely related to human activities, and multiomics still revealed the potential public risk to human health.

Comparison of virulence-related determinants between the ST59-t437 and ST239-t030 genotypes of methicillin-resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen for human infection. Hospital-acquired (HA) and community-acquired (CA) MRSA infections are serious clinical problems worldwide. In this study, we selected typical HA-MRSA strain and CA-MRSA isolates from our previous research and compared their phenotypic and pathogenic abilities both in vitro and in vivo. RESULTS: ST59-t437-SCCmecIVa (YNSA7) and ST59-t437-SCCmecVb (YNSA53) belonged to two prevalent subclones of CA-MRSA, while ST239-t030-SCCmecIII (YNSA163) was an HA-MRSA epidemic clone in Southwest China. ST59-t437 strains demonstrated faster growth ability, higher survival rate resistance to human blood, and more toxin secretion levels and cytotoxicity than ST239-t030. The virulence and regulatory genes of hld, psm-α, RNAIII, agrA, and crtN were highly expressed on CA-MRSA isolates, especially the ST59-t437-SCCmecIVa subclone. However, the ST239-t030 strain had the strongest adhesion and biofilm ability among these MRSA bacteria. Animal experiments revealed the most serious lethal effect on BALB/c mice caused by the YNSA7 strain infection. The survival rates of BALB/c mice infected with the three MRSA strains were 16.7, 50.0 and 100.0% for YNSA7, YNSA53 and YNSA163, respectively. Histopathological analyses of infected animals indicated that the lungs were the most seriously damaged organs, especially for ST59-t437 MRSA. Severe inflammatory reactions, tissue destruction, and massive exudation of inflammatory mediators and cells could be identified in ST59-t437 strain-infected animals. CONCLUSIONS: In general, ST59-t437 strains showed higher pathogenic ability than the ST239-t030 isolate, while ST239-t030 MRSA revealed the features prevalent in hospital settings, specifically for adhesion and biofilm ability.

Response of the gut microbiota during the Clostridioides difficile infection in tree shrews mimics those in humans.

BACKGROUND: Clostridioides difficile is a major cause of antibiotic associated diarrhea. Several animal models are used to study C. difficile infection (CDI). The tree shrew has recently been developed as a model of primate processes. C. difficile infection has not been examined in tree shrews. We infected tree shrews with hyper-virulent C. difficile strains and examined the alterations in gut microbiota using 16S rRNA gene sequencing. RESULTS: C. difficile colonized the gastrointestinal tract of tree shrew and caused diarrhea and weight loss. Histopathologic examination indicated structures and mucosal cell destruction in ileal and colonic tissues. The gut microbial community was highly diversity before infection and was dominated by Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria. Antibiotic administration decreased the diversity of the gut microbiota and led to an outgrowth of Lactobacillus. The relative abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Lachnospiraceae, Enterobacteriaceae, Escherichia, Blautia, and Tyzzerella increased following C. difficile infection. These taxa could be biomarkers for C. difficile colonization. CONCLUSIONS: In general, the disease symptoms, histopathology, and gut microbiota changes following C. difficile infection in tree shrews were similar to those observed in humans.

The molecular characters and antibiotic resistance of Clostridioides difficile from economic animals in China.

BACKGROUND: It has been performed worldwidely to explore the potential of animals that might be a reservoir for community associated human infections of Clostridioides difficile. Several genetically undistinguished PCR ribotypes of C. difficile from animals and human have been reported, illustrating potential transmission of C. difficile between them. Pig and calf were considered as the main origins of C. difficile with predominant RT078 and RT033, respectively. As more investigations involved, great diversity of molecular types from pig and calf were reported in Europe, North American and Australia. However, there were quite limited research on C. difficile isolates from meat animals in China, leading to non-comprehensive understanding of molecular epidemiology of C. difficile in China. RESULTS: A total of 55 C. difficile were isolated from 953 animal stool samples, within which 51 strains were from newborn dairy calf less than 7 days in Shandong Province. These isolates were divided into 3 STs and 6 RTs, of which ST11/RT126 was predominant type, and responsible for majority antibiotic resistance isolates. All the isolates were resistant to at least one tested antibiotics, however, only two multidrug resistant (MDR) isolates were identified. Furthermore, erythromycin (ERY) and clindamycin (CLI) were the two main resistant antibiotics. None of the isolates were resistant to vancomycin (VAN), metronidazole (MTZ), tetracycline (TET), and rifampin (RIF). CONCLUSIONS: In this study, we analyzed the prevalence, molecular characters and antibiotic resistance of C. difficile from calf, sheep, chicken, and pig in China. Some unique features were found here: first, RT126 not RT078 were the dominant type from baby calf, and none isolates were got from pig; second, on the whole, isolates from animals display relative lower resistant rate to these 11 tested antibiotics, compared with isolates from human in China in our previous report. Our study helps to deep understanding the situation of C. difficile from economic animals in China, and to further study the potential transmission of C. difficile between meat animals and human.

A comparative genomic analysis between methicillin-resistant Staphylococcus aureus strains of hospital acquired and community infections in Yunnan province of China.

BACKGROUND: Currently, Staphylococcus aureus is one of the most important pathogens worldwide, especially for methicillin-resistant S. aureus (MRSA) infection. However, few reports referred to patients' MRSA infections in Yunnan province, southwest China. METHODS: In this study, we selected representative MRSA strains from patients' systemic surveillance in Yunnan province of China, performed the genomic sequencing and compared their features, together with some food derived strains. RESULTS: Among sixty selective isolates, forty strains were isolated from patients, and twenty isolated from food. Among the patients' strains, sixteen were recognized as community-acquired (CA), compared with 24 for hospital-acquired (HA). ST6-t701, ST59-t437 and ST239-t030 were the three major genotype profiles. ST6-t701 was predominated in food strains, while ST59-t437 and ST239-t030 were the primary clones in patients. The clinical features between CA and HA-MRSA of patients were statistical different. Compared the antibiotic resistant results between patients and food indicated that higher antibiotic resistant rates were found in patients' strains. Totally, the average genome sizes of 60 isolates were 2.79 ± 0.05 Mbp, with GC content 33% and 84.50 ± 0.20% of coding rate. The core genomes of these isolates were 1593 genes. Phylogenetic analysis based on pan-genome and SNP of strains showed that five clustering groups were generated. Clustering ST239-t030 contained all the HA-MRSA cases in this study; clustering ST6-t701 referred to food and CA-MRSA infections in community; clustering ST59-t437 showed the heterogeneity for provoking different clinical diseases in both community and hospital. Phylogenetic tree, incorporating 24 isolates from different regions, indicated ST239-t030 strains in this study were more closely related to T0131 isolate from Tianjin, China, belonged to 'Turkish clade' from Eastern Europe; two groups of ST59-t437 clones of MRSA in Yunnan province were generated, belonged to the 'Asian-Pacific' clone (AP) and 'Taiwan' clone (TW) respectively. CONCLUSIONS: ST239-t030, ST59-t437 and ST6-t701 were the three major MRSA clones in Yunnan province of China. ST239-t030 clonal Yunnan isolates demonstrated the local endemic of clone establishment for a number of years, whereas ST59-t437 strains revealed the multi-origins of this clone. In general, genomic study on epidemic clones of MRSA in southwest China provided the features and evolution of this pathogen.

The characteristics of gut microbiota and commensal Enterobacteriaceae isolates in tree shrew (Tupaia belangeri).

BACKGROUND: Tree shrew is a novel laboratory animal with specific characters for human disease researches in recent years. However, little is known about its characteristics of gut microbial community and intestinal commensal bacteria. In this study, 16S rRNA sequencing method was used to illustrate the gut microbiota structure and commensal Enterobacteriaceae bacteria were isolated to demonstrate their features. RESULTS: The results showed Epsilonbacteraeota (30%), Proteobacteria (25%), Firmicutes (19%), Fusobacteria (13%), and Bacteroidetes (8%) were the most abundant phyla in the gut of tree shrew. Campylobacteria, Campylobacterales, Helicobacteraceae and Helicobacter were the predominant abundance for class, order, family and genus levels respectively. The alpha diversity analysis showed statistical significance (P < 0.05) for operational taxonomic units (OTUs), the richness estimates, and diversity indices for age groups of tree shrew. Beta diversity revealed the significant difference (P < 0.05) between age groups, which showed high abundance of Epsilonbacteraeota and Spirochaetes in infant group, Proteobacteria in young group, Fusobacteria in middle group, and Firmicutes in senile group. The diversity of microbial community was increased followed by the aging process of this animal. 16S rRNA gene functional prediction indicated that highly hot spots for infectious diseases, and neurodegenerative diseases in low age group of tree shrew (infant and young). The most isolated commensal Enterobacteriaceae bacteria from tree shrew were Proteus spp. (67%) and Escherichia coli (25%). Among these strains, the antibiotic resistant isolates were commonly found, and pulsed-field gel electrophoresis (PFGE) results of Proteus spp. indicated a high degree of similarity between isolates in the same age group, which was not observed for other bacteria. CONCLUSIONS: In general, this study made understandings of the gut community structure and diversity of tree shrew.

Molecular characteristics of Staphylococcus aureus isolates from food surveillance in southwest China.

BACKGROUND: Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases in the world. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and spa typing methods were used to characterize Staphylococcus aureus isolates from food surveillance during 2013-2015 in southwest China, and Staphylococcal cassette chromosome mec (SCCmec) typing was used for methicillin-resistant S. aureus (MRSA). Isolates were also examined for their antibiotic resistance and carriage of virulence genes. RESULTS: Isolation rate of S. aureus was 2.60% during the three years' surveillance and 29.50% of them were MRSA. All the S. aureus had hla genes (100%), 14.34% of the strains had tst, and 16.73% had PVL. 163 PFGE-SmaI patterns, 41 ST types and 36 spa types were obtained for all the S. aureus. Among them, ST6-t701 (13.15%), ST7-t091 (12.75%), ST59-t437 (9.96%) and ST5-t002 (7.57%) were the prevalent genotypes. Most of MRSA in this study belonged to SCCmec IV and V, accounted for 74.32% and 20.27% respectively. ST6-SCCmec IV-t701 (36.50%) was the most prevalent clone among isolates from food, followed by ST59-SCCmec V-t437 (20.30%), ST5-SCCmec IV-t002 (12.20%) and ST59-SCCmec IV-t437 (12.20%). Some strains had the identical PFGE patterns, ST and spa types with isolates from patients. CONCLUSIONS: S. aureus isolated from food in southwest China displayed heterogeneity. Isolates had the same genotype profiles with isolates from patients, indicating high homology.

Complete genome sequence and comparative genome analysis of a new special Yersinia enterocolitica.

Yersinia enterocolitica is the most diverse species among the Yersinia genera and shows more polymorphism, especially for the non-pathogenic strains. Individual non-pathogenic Y. enterocolitica strains are wrongly identified because of atypical phenotypes. In this study, we isolated an unusual Y. enterocolitica strain LC20 from Rattus norvegicus. The strain did not utilize urea and could not be classified as the biotype. API 20E identified Escherichia coli; however, it grew well at 25 °C, but E. coli grew well at 37 °C. We analyzed the genome of LC20 and found the whole chromosome of LC20 was collinear with Y. enterocolitica 8081, and the urease gene did not exist on the genome which is consistent with the result of API 20E. Also, the 16 S and 23 SrRNA gene of LC20 lay on a branch of Y. enterocolitica. Furthermore, the core-based and pan-based phylogenetic trees showed that LC20 was classified into the Y. enterocolitica cluster. Two plasmids (80 and 50 k) from LC20 shared low genetic homology with pYV from the Yersinia genus, one was an ancestral Yersinia plasmid and the other was novel encoding a number of transposases. Some pathogenic and non-pathogenic Y. enterocolitica-specific genes coexisted in LC20. Thus, although it could not be classified into any Y. enterocolitica biotype due to its special biochemical metabolism, we concluded the LC20 was a Y. enterocolitica strain because its genome was similar to other Y. enterocolitica and it might be a strain with many mutations and combinations emerging in the processes of its evolution.

Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein.

BACKGROUND: Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains. RESULTS: The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar. CONCLUSIONS: OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.

Homology analysis of pathogenic Yersinia species Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis based on multilocus sequence typing.

We developed a multilocus sequence typing (MLST) scheme and used it to study the population structure and evolutionary relationships of three pathogenic Yersinia species. MLST of these three Yersinia species showed a complex of two clusters, one composed of Yersinia pseudotuberculosis and Yersinia pestis and the other composed of Yersinia enterocolitica. Within the first cluster, the predominant Y. pestis sequence type 90 (ST90) was linked to Y. pseudotuberculosis ST43 by one locus difference, and 81.25% of the ST43 strains were from serotype O:1b, supporting the hypothesis that Y. pestis descended from the O:1b serotype of Y. pseudotuberculosis. We also found that the worldwide-prevalent serotypes O:1a, O:1b, and O:3 were predominated by specific STs. The second cluster consisted of pathogenic and nonpathogenic Y. enterocolitica strains, two of which may not have identical STs. The pathogenic Y. enterocolitica strains formed a relatively conserved group; most strains clustered within ST186 and ST187. Serotypes O:3, O:8, and O:9 were separated into three distinct blocks. Nonpathogenic Y. enterocolitica STs were more heterogeneous, reflecting genetic diversity through evolution. By providing a better and effective MLST procedure for use with the Yersinia community, valuable information and insights into the genetic evolutionary differences of these pathogens were obtained.

Comparison of cytokine immune responses to Brucella abortus and Yersinia enterocolitica serotype O:9 infections in BALB/c mice.

Brucella abortus and Yersinia enterocolitica serotype O:9 serologically cross-react in the immune response with the host; therefore, our aim was to compare the immune responses to these two pathogens. We selected typical B. abortus and Y. enterocolitica O:9 strains to study the cytokine immune response and the histopathological changes in livers and spleens of BALB/c mice. The data showed the cytokine responses to the two strains of pathogens were different, where the average levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-α) were higher with B. abortus infections than with Y. enterocolitica O:9 infections, especially for IFN-γ, while the IL-10 level was lower and the levels of IL-1ß, IL-4, IL-5, and IL-6 were similar. The histopathological effects in the livers and spleens of the BALB/c mice with B. abortus and Y. enterocolitica O:9 infections were similar; however, the pathological changes in the liver were greater with B. abortus infections, while damage in the spleen was greater with Y. enterocolitica O:9 infections. These observations show that different cytokine responses and histopathological changes occur with B. abortus and Y. enterocolitica O:9 infections.

Comparative analysis of shotgun metagenomics and 16S rDNA sequencing of gut microbiota in migratory seagulls.

Background: Shotgun metagenomic and 16S rDNA sequencing are commonly used methods to identify the taxonomic composition of microbial communities. Previously, we analysed the gut microbiota and intestinal pathogenic bacteria configuration of migratory seagulls by using 16S rDNA sequencing and culture methods. Methods: To continue in-depth research on the gut microbiome and reveal the applicability of the two methods, we compared the metagenome and 16S rDNA amplicon results to further demonstrate the features of this animal. Results: The number of bacterial species detected by metagenomics gradually increased from the phylum to species level, consistent with 16S rDNA sequencing. Several taxa were commonly shared by both sequencing methods. However, Escherichia, Shigella, Erwinia, Klebsiella, Salmonella, Escherichia albertii, Shigella sonnei, Salmonella enterica, and Shigella flexneri were unique taxa for the metagenome compared with Escherichia-Shigella, Hafnia-Obesumbacterium, Catellicoccus marimammalium, Lactococcus garvieae, and Streptococcus gallolyticus for 16S rDNA sequencing. The largest differences in relative abundance between the two methods were identified at the species level, which identified many pathogenic bacteria to humans using metagenomic sequencing. Pearson correlation analysis indicated that the correlation coefficient for the two methods gradually decreased with the refinement of the taxonomic levels. The high consistency of the correlation coefficient was identified at the genus level for the beta diversity of the two methods. Conclusions: In general, relatively consistent patterns and reliability could be identified by both sequencing methods, but the results varied following the refinement of taxonomic levels. Metagenomic sequencing was more suitable for the discovery and detection of pathogenic bacteria of gut microbiota in seagulls. Although there were large differences in the numbers and abundance of bacterial species of the two methods in terms of taxonomic levels, the patterns and reliability results of the samples were consistent.

Prevalence of Yersinia enterocolitica in pigs slaughtered in Chinese abattoirs.

The distribution of Yersinia enterocolitica in slaughtered pigs in China was studied. A total of 8,773 samples were collected and examined from different pig abattoirs in 11 provinces from 2009 to 2011. Of these, 4,495 were oral-pharyngeal swab (tonsils) samples from pigs, 1,239 were from intestinal contents, and 3,039 were feces samples from abattoirs or local pigpens. The data showed that 1,132 strains were obtained, from which the isolation rate for Yersinia enterocolitica was 19.53% (878/4,495) from the tonsil samples, 7.51% (93/1,239) from intestinal contents, and 5.30% (161/3,039) from feces. Of the 850 pathogenic Yersinia strains, except for three of bioserotype 2/O:9 and three of bioserotype 4/O:3, most (844/850) were of bioserotype 3/O:3. Interestingly, pathogenic Y. enterocolitica accounted for the majority of the isolated strains from most provinces (85.17% to 100%), whereas from Heilongjiang, 96.52% (111/115) were classified as nonpathogenic biotype 1A with various serotypes, and only 3.48% of the strains (4/115) were pathogenic 3/O:3. All of the pathogenic strains were analyzed using pulsed-field gel electrophoresis (PFGE), and 49 patterns were obtained for the O:3 pathogenic strains; most of them were K6GN11C30021 (53.13%: 450/847) and K6GN11C30012 (21.37%: 181/847). Several strains from diarrhea patient samples revealed PFGE patterns identical to that from samples of local pigs, suggesting a possible link between porcine isolates and human infection. The results above suggested that Yersinia enterocolitica in slaughtered pigs from Chinese abattoirs was characterized by region-specific PFGE patterns and confirmed that strains isolated from pigs are closely related to those from human infections.

Comparative antigenic proteins and proteomics of pathogenic Yersinia enterocolitica bio-serotypes 1B/O: 8 and 2/O: 9 cultured at 25°C and 37°C.

Yersinia enterocolitica is a Gram-negative enteric pathogen responsible for a number of gastrointestinal disorders; the most pathogenic bio-serotype is 1B/O: 8. In this study, we compared the antigenicity of the outer membrane proteins and proteomics of the whole-cell proteins of a pathogenic bio-serotype 2/O: 9 isolated in China and a bio-serotype 1B/O: 8 strain isolated in Japan. Using two-dimensional gel electrophoresis, we showed that the outer membrane proteins A (OmpA), C (OmpC) and F (OmpF) were the major antigens for both strains, although proteins located on the bacterial cell membrane and enzymes involved in energy metabolism were also identified as antigenic. We compared the whole-cell proteins of the two strains cultured at 25°C and 37°C and found portions of the outer membrane proteins (OmpX, OmpF and OmpA) were downregulated when the bacteria were cultured at 37°C, whereas urease subunit gamma (UreA), urease subunit alpha (UreC) and urease accessory protein (UreE), which are involved in urease synthesis, were upregulated when the bacteria were grown at 37°C. These observations will lay a foundation to selection of diagnostic markers for pathogenic Yersinia enterocolitica, and maybe contribute to choose the vaccine targets.

Antibiotic resistance and genomic features of Clostridioides difficile in southwest China.

Background: Clostridioides difficile infection (CDI) caused by toxigenic strains leads to antibiotic-related diarrhea, colitis, or even fatal pseudomembranous enteritis. Previously, we conducted a cross-sectional study on prevalence of CDI in southwest China. However, the antibiotics resistance and characteristics of genomes of these isolates are still unknown. Methods: Antibiotic susceptibility testing with E-test strips and whole genome sequence analysis were used to characterize the features of these C. difficile isolates. Results: Forty-nine strains of C. difficile were used in this study. Five isolates were non-toxigenic and the rest carried toxigenic genes. We have previously reported that ST35/RT046, ST3/RT001 and ST3/RT009 were the mostly distributed genotypes of strains in the children group. In this study, all the C. difficile isolates were sensitive to metronidazole, meropenem, amoxicillin/clavulanic acid and vancomycin. Most of the strains were resistant to erythromycin, gentamicin and clindamycin. The annotated resistant genes, such as macB, vanRA, vanRG, vanRM, arlR, and efrB were mostly identified related to macrolide, glycopeptide, and fluoroquinolone resistance. Interestingly, 77.55% of the strains were considered as multi-drug resistant (MDR). Phylogenetic analysis based on core genome of bacteria revealed all the strains were divided into clade 1 and clade 4. The characteristics of genome diversity for clade 1 could be found. None of the isolates showed 18-bp deletion of tcdC as RT027 strain as described before, and polymorphism of tcdB showed a high degree of conservation than tcdA gene. Conclusions: Most of the C. difficile isolates in this study were resistant to macrolide and aminoglycoside antibiotics. Moreover, the MDR strains were commonly found. All the isolates belonged to clade 1 and clade 4 according to phylogenetic analysis of bacterial genome, and highly genomic diversity of clade 1 was identified for these strains.

An imported human case with the SARS-CoV-2 Omicron subvariant BA.2.75 in Yunnan Province, China.

The Omicron variants spread rapidly worldwide after being initially detected in South Africa in November 2021. It showed increased transmissibility and immune evasion with far more amino acid mutations in the spike (S) protein than the previously circulating variants of concern (VOCs). Notably, on 15 July 2022, we monitored the first VOC / Omicron subvariant BA.2.75 in China from an imported case. Moreover, nowadays, this subvariant still is predominant in India. It has nine additional mutations in the S protein compared to BA.2, three of which (W152R, G446S, and R493Q reversion) might contribute to higher transmissibility and immune escape. This subvariant could cause wider spread and pose a threat to the global situation. Our timely reporting and continuous genomic analysis are essential to fully elucidate the characteristics of the subvariant BA.2.75 in the future.

miR-204-3p downregulates KRT16 and promotes corneal repair in tree shrew fungal keratitis model.

OBJECTIVES: Corneal repair is critical for the treatment and recovery of corneal injuries. However, the molecular mechanism underlying corneal repair remains unclear. METHODS: A tree shrew model of corneal fungal infection was established by injecting Fusarium solani into the corneal stroma to study the role of miR-204-3p in repairing corneal injury induced by fungal keratitis and to explore the potential mechanisms underlying the repair process. RESULTS: miR-204-3p expression was significantly downregulated, while KRT16 expression was significantly upregulated after F. solani infection in the cornea of tree shrews. Moreover, miR-204-3p injection promoted corneal injury repair post-infection, potentially by downregulating KRT16 expression. Results of a luciferase reporter gene assay showed that miR-204-3p had a targeted relationship with KRT16. KRT16 protein expression levels decreased after miR-204-3p injection into the cornea with fungal keratitis, reducing the degree of corneal injury. CONCLUSIONS: In this study, we report for the first time that miR-204-3p and KRT16 influence the repair of corneal injury. In addition, their effects on the repair of corneal injury were studied in a tree shrew model, providing an experimental basis for the study of pathogenesis of human fungal keratitis.

Community-acquired methicillin-resistant Staphylococcus aureus provoked cytokine storm causing severe infection on BALB/c mice.

Methicillin-resistant Staphylococcus aureus (MRSA) has become the most important pathogen of hospital-acquired (HA) or community-acquired (CA) infections. However, it is unclear of the cytokines responsible for pathological hyper-inflammation in sepsis related cytokine storm for MRSA infection. In this study, we selected typical HA-MRSA strain (YNSA163: ST239-t030-SCCmecⅢ) and two CA-MRSA isolates (YNSA7: ST59-t439-SCCmecⅣa and YNSA53: ST59-t437-SCCmecⅤb) from our previous research, infected on BALB/c mice, and analyzed the cytokine storm patterns during infection process. The animal experiments revealed the most serious lethal effect on BALB/c mice caused by YNSA7 strain infection, followed by YNSA53, and no BALB/c mice died for YNSA163 infection. Histopathological analyses revealed that lung was the most seriously damaged organs, followed by spleen and kidney, especially for CA-MRSA infection. The severe inflammatory reactions, tissue destruction, and massive exudation of inflammatory mediators and cells could be identified in CA-MRSA strains infected mice. Interleukin-6 (IL-6) and IL-10 were both highly expressed in spleen and lung of YNSA7 and YNSA53 dead cases compared with YNSA53 survived and YNSA163 cases, which demonstrated cytokine storm pattern for CA-MRSA strains infection. The results of IL-6 intervention experiment verified that the enhanced IL-6 secretion was responsible for the host lethality of YNSA7 infection. RNA-sequencing results among three MRSA isolates indicated most of the differentially expressed genes referred to cellular process, metabolism and genetic information processing of bacteria. Specifically, clpP, chp chemotaxis inhibit, fnbB, pathogencity island protein and virulence associated protein E were highly expressed in YNSA7 strain. In general, CA-MRSA strains provoked cytokine storm on BALB/c mice led to severe infection and lethality, the up-regulated of some virulence genes might play important role in pathogenesis.

Characteristics of Angiotensin I-converting enzyme 2, type II transmembrane serine protease 2 and 4 in tree shrew indicate it as a potential animal model for SARS-CoV-2 infection.

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.