Novel Streptococcus uberis sequence types causing bovine subclinical mastitis in Hainan, China.

AIM: To determine the molecular epidemiology, genotypes and phenotypes of the major species of Streptococcus associated with bovine subclinical mastitis in Hainan, China. METHODS AND RESULTS: In total, 150 subclinical mastitis milk samples were collected from two large dairy farms in Hainan. On the basis of biochemical tests and 16S rDNA sequencing, 39 samples were Streptococcus positive and the most frequently isolated species was Streptococcus uberis (n = 29, 74.4%). According to multilocus sequence typing (MLST), and assays of biofilm formation, antimicrobial susceptibility, resistance and virulence genes, the S. uberis isolates were clustered into nine new sequence types (STs; ST986-ST994) but were not merged into a clonal group (except for ST991 [CC143]). All isolates produced biofilm, but most weakly. The dominant virulence pattern was hasABC + sua + gapC + oppF + pauA + mtuA + cfu (27/29, 91.1%), based on the 11 virulence genes tested. The majority of isolates (88.46%) carried at least one resistance gene, and more than half (58.62%) were multidrug-resistant. The main resistance genes were linB (65.5%), ermB (37.9%) and tetS (34.5%), among the six antibiotic resistance genes and 11 antimicrobials tested. CONCLUSION: Environmental S. uberis is important in bovine subclinical mastitis in Hainan. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus uberis isolates in Hainan, China, show distinct MLST, virulence and antibiotic resistance characteristics.

Genome-wide association study identifies 7q11.22 and 7q36.3 associated with noise-induced hearing loss among Chinese population.

Noise-induced hearing loss (NIHL) seriously affects the life quality of humans and causes huge economic losses to society. To identify novel genetic loci involved in NIHL, we conducted a genome-wide association study (GWAS) for this symptom in Chinese populations. GWAS scan was performed in 89 NIHL subjects (cases) and 209 subjects with normal hearing who have been exposed to a similar noise environment (controls), followed by a replication study consisting of 53 cases and 360 controls. We identified that four candidate pathways were nominally significantly associated with NIHL, including the Erbb, Wnt, hedgehog and intraflagellar transport pathways. In addition, two novel index single-nucleotide polymorphisms, rs35075890 in the intron of AUTS2 gene at 7q11.22 (combined P = 1.3 × 10-6 ) and rs10081191 in the intron of PTPRN2 gene at 7q36.3 (combined P = 2.1 × 10-6 ), were significantly associated with NIHL. Furthermore, the expression quantitative trait loci analyses revealed that in brain tissues, the genotypes of rs35075890 are significantly associated with the expression levels of AUTS2, and the genotypes of rs10081191 are significantly associated with the expressions of PTPRN2 and WDR60. In conclusion, our findings highlight two novel loci at 7q11.22 and 7q36.3 conferring susceptibility to NIHL.

A double-locus scarless genome editing system in Escherichia coli.

OBJECTIVE: To develop a convenient double-locus scarless genome editing system in Escherichia coli, based on the type II Streptococcus pyogenes CRISPR/Cas9 and λ Red recombination cassette. RESULTS: A two-plasmid genome editing system was constructed. The large-sized plasmid harbors the cas9 and λ Red recombination genes (gam, bet, and exo), while the small-molecular plasmid can simultaneously express two different gRNAs (targeting genome RNAs). The recombination efficiency was tested by targeting the galK, lacZ, and dbpA genes in E. coli with ssDNA or dsDNA. Resulting concurrent double-locus recombination efficiencies were 88 ± 5.5% (point mutation), 39.7 ± 4.3% (deletion/insertion), and 57.8 ± 3.4%-58.5 ± 4.1% (mixed point and deletion/insertion mutation), depending on 30 (ssDNA) or 40 bp (dsDNA) homologous side arms employed. In addition, the curing efficiency of the guide plasmid expressing gRNAs for negative selection was higher (96 ± 3% in 4 h) than the help plasmid carrying cas9 and λ Red (92 ± 2% in 9 h). CONCLUSIONS: The new editing system is convenient and efficient for simultaneous double-locus recombination in the genome and should be favorable for high-throughput multiplex genome editing in synthetic biology and metabolic engineering.

An outbreak of Providencia rettgeri bacteremia at a Ptyas mucosus farm in Hainan, China.

Aim: To describe the histopathology and etiology of an outbreak of respiratory disease at a Ptyas mucosus farm in Hainan, China. Methods and results: The etiology was confirmed by gross examination and microscopic analysis. The bacterial isolates from blood and internal organs were identified by biochemical analysis and 16S rRNA gene sequencing. The virulence and antibiotic resistance characteristics of the isolates were further demonstrated by polymerase chain reaction (PCR), disk diffusion testing, and LD50 analysis in Kunming mice. Histopathological analysis of the diseased P. mucosus revealed systemic lesions, including severe airway obstruction with large numbers of inflammatory cells and cellulose exudates in the lungs; severe multifocal hepatocyte vacuolar degeneration and necrosis in the liver with excessive inflammatory exudates and chronic granuloma; splenic hemorrhage and partial loss of splenic structure; and renal vascular and interstitial congestion. Providencia rettgeri was isolated from the blood and multiple internal organs (liver, spleen, kidneys, and lungs). All examined isolates (H1, H4, and H13) were multidrug-resistant but sensitive to four antibiotics-cefepime, imipenem, chloramphenicol, and ciprofloxacin. Both H1 and H4 carried five resistance genes [bla OXA, tet(A), tet(B), tet(E), and aac (3)-IIa], whereas H13 only carried the tet(A) gene. The dominant virulence pattern of the three isolates was hlyA + ZapA + luxS + rsbA. The virulence of H1 strain was tested, and its 50% lethal dose (LD50) in mice was 2.29 × 108 CFU ml-1. Conclusion: To our knowledge, this is the first study to describe an outbreak of bacteremia caused by P. rettgeri in farmed rat snakes. Significance and impact of the study: The results highlight that P. rettgeri is an emerging bacterial pathogen in farmed reptiles.

Complete genome sequence analysis of Edwardsiella tarda SC002 from hatchlings of Siamese crocodile.

Edwardsiella tarda is a Gram-negative, facultative anaerobic rod-shaped bacterium and the causative agent of the systemic disease "Edwardsiellosis". It is commonly prevalent in aquatic organisms with subsequent economic loss and hence has attracted increasing attention from researchers. In this study, we investigated the complete genome sequence of a highly virulent isolate Edwardsiella tarda SC002 isolated from hatchlings of the Siamese crocodile. The genome of SC002 consisted of one circular chromosome of length 3,662,469 bp with a 57.29% G+C content and four novel plasmids. A total of 3,734 protein-coding genes, 12 genomic islands (GIs), 7 prophages, 48 interspersed repeat sequences, 248 tandem repeat sequences, a CRISPR component with a total length of 175 bp, and 171 ncRNAs (tRNA = 106, sRNA = 37, and rRNA = 28) were predicted. In addition, the coding genes of assembled genome were successfully annotated against eight general databases (NR = 3,618/3,734, COG = 2,947/3,734, KEGG = 3,485/3,734, SWISS-PROT = 2,787/3,734, GO = 2,648/3,734, Pfam = 2,648/3,734, CAZy = 130/3,734, and TCDB = 637/3,734) and four pathogenicity-related databases (ARDB = 11/3,734, CARD = 142/3,734, PHI = 538/3,734, and VFDB = 315/3,734). Pan-genome and comparative genome analyses of the complete sequenced genomes confirmed their evolutionary relationships. The present study confirmed that E. tarda SC002 is a potential pathogen bearing a bulk amount of antibiotic resistance, virulence, and pathogenic genes and its open pan-genome may enhance its host range in the future.

A nosocomial Pseudomonas aeruginosa ST3495 isolated from a wild Burmese python (Python bivittatus) with suppurative pneumonia and bacteremia in Hainan, China.

Pseudomonas aeruginosa is a common infectious agent associated with respiratory diseases in boas and pythons, however, the histopathology, resistance and virulence are yet described for this species. In this study, we investigated a dying Burmese python rescued from tropical rainforest in Hainan. Clinical signs were open-mouthed breathing, abnormal shedding and anorexia. Abundant yellow mucopurulent secretions were observed in highly ectatic segmental bronchi by postmortem. Histopathological lesions included systemic pneumonia, enteritis, nephritis and carditis. P. aeruginosa was the only species isolated from heart blood, kidney, trachea and lung. The phenotype analysis demonstrated that the isolates had strong biofilm, and were sensitive to amikacin, spectinomycin, ciprofloxacin, norfloxacin and polymyxin B, moreover, the LD50 of the most virulent isolate was 2.22×105 cfu/mL in a zebrafish model. Molecular epidemiological analysis revealed that the isolates belonged to sequence type 3495, the common gene patterns were toxA + exoSYT + phzIM + plcHN in virulence and catB + blaTEM + ant (3'')-I+ tetA in resistance. This study highlights that P. aeruginosa should be worth more attention in wildlife conservation and raise the public awareness for the cross infection and cross spread between animals and human.

Quantitative proteomics identifies a plasma multi-protein model for detection of hepatocellular carcinoma.

More efficient biomarkers are needed to facilitate the early detection of hepatocellular carcinoma (HCC). We aimed to identify candidate biomarkers for HCC detection by proteomic analysis. First, we performed a global proteomic analysis of 10 paired HCC and non-tumor tissues. Then, we validated the top-ranked proteins by targeted proteomic analyses in another tissue cohort. At last, we used enzyme-linked immunosorbent assays to validate the candidate biomarkers in multiple serum cohorts including HCC cases (HCCs), cirrhosis cases (LCs), and normal controls (NCs). We identified and validated 33 up-regulated proteins in HCC tissues. Among them, eight secretory or membrane proteins were further evaluated in serum, revealing that aldo-keto reductase family 1 member B10 (AKR1B10) and cathepsin A (CTSA) can distinguish HCCs from LCs and NCs. The area under the curves (AUCs) were 0.891 and 0.894 for AKR1B10 and CTSA, respectively, greater than that of alpha-fetoprotein (AFP; 0.831). Notably, combining the three proteins reached an AUC of 0.969, which outperformed AFP alone (P < 0.05). Furthermore, the serum AKR1B10 levels dramatically decreased after surgery. AKR1B10 and CTSA are potential serum biomarkers for HCC detection. The combination of AKR1B10, CTSA, and AFP may improve the HCC diagnostic efficacy.

Identification of a Novel Ichthyic Parvovirus in Marine Species in Hainan Island, China.

Parvoviruses are a diverse group of viruses that are capable of infecting a wide range of animals. In this study, we report the discovery of a novel parvovirus, tilapia parvovirus HMU-HKU, in the fecal samples of crocodiles and intestines of tilapia in Hainan Province, China. The novel parvovirus was firstly identified from crocodiles fed with tilapia using next-generation sequencing (NGS). Screening studies revealed that the prevalence of the novel parvovirus in crocodile feces samples fed on tilapia (75-86%) was apparently higher than that in crocodiles fed with chicken (4%). Further studies revealed that the prevalence of the novel parvovirus in tilapia feces samples collected at four areas in Hainan Province was between 40 and 90%. Four stains of the novel parvovirus were identified in this study based on sequence analyses of NS1 and all the four strains were found in tilapia in contrast only two of them were detected in crocodile feces. The nearly full-length genome sequence of the tilapia parvovirus HMU-HKU-1 was determined and showed less than 45.50 and 40.38% amino acid identity with other members of Parvoviridae in NS1 and VP1 genes, respectively. Phylogenetic analysis based on the complete helicase domain amino acid sequences showed that the tilapia parvovirus HMU-HKU-1 formed a relatively independent branch in the newly proposed genus Chaphamaparvovirus in the subfamily Hamaparvovirinae according to the ICTV's most recent taxonomic criteria for Parvoviridae classification. Tilapia parvovirus HMU-HKU-1 likely represented a new species within the new genus Chaphamaparvovirus. The identification of tilapia parvovirus HMU-HKU provides further insight into the viral and genetic diversity of parvoviruses and its infections in tilapia populations need to be evaluated in terms of pathogenicity and production losses in tilapia farming.