Nocardia implantans sp. nov. isolated from a patient with pulmonary infection.

TwoGram-stain-positive and rod-shaped actinomycetes (strains CDC186T and CDC192) were isolated from sputum samples of a patient in Chongqing, PR China, and were investigated to determine their taxonomic status. The results of phylogenetic analysis based on the 16S rRNA gene indicated that CDC186T and CDC192 represented members of the genus Nocardia, and the sequence similarity with Nocardia beijingensis DSM 44636T was the highest, at 99.71 and 99.78 %, respectively. The DNA G+C content of both CDC186T and CDC192 was 69.1 %. Genomic diversity analysis revealed that the average nucleotide identity and in silico DNA‒DNA hybridisation values between the two novel strains and closely related species were significantly below the thresholds of 95-96 and 70 %, respectively, but these values between the two novel strains were 99.96 and 99.90 %, respectively. The phylogenetic relationship based on the dapb1 gene and the single-copy core genes further indicated that the two novel strains were clustered in separate branch adjacent to N. beijingensis DSM 44636T. Growth occurred within the ranges of 20-42 °C, pH 6.0-9.0 and NaCl concentrations of 0.5-4.5 % (w/v). The major fatty acids of CDC186T and CDC192 were C16 : 0 and C18 : 0 10-methyl [tuberculostearic acid (TBSA)]. The predominant respiratory menaquinone was MK-9. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, one unidentified glycolipid, one unidentified phospholipid and one unidentified phosphoglycolipid. All the genomes of the studied strains were annotated with virulence factor (VF)-associated genes homologous to those of Mycobacterium tuberculosis, and the results of susceptibility testing indicated that CDC186T and CDC192 were resistant to amoxicillin-clavulanic acid and tigecycline. On the basis of chemotaxonomic characteristics and the results of phylogenetic analyses, strains CDC186T and CDC192 represent a novel species within the genus Nocardia, for which the name Nocardia implantans sp. nov. is proposed. The type strain is CDC186T (=GDMCC 4.206T= JCM 34959T).

Paeoniflorin alleviates inflammation in bovine mammary epithelial cells induced by Staphylococcus haemolyticus through TLR2/NF-κB signaling pathways.

Staphylococcus haemolyticus (S. haemolyticus) is one of the most common coagulase-negative staphylococci (CoNS) isolates from bovine mastitis. Paeoniflorin (PF) shows anti-inflammatory effects on different inflammatory diseases in vitro studies and in vivo animal experiments. In this study, the viability of bovine mammary epithelial cells (bMECs) was detected by the cell counting kit-8 experiment. Subsequently, bMECs were induced with S. haemolyticus, and the induction dosage was determined. The expression of pro-inflammatory cytokines and toll-like receptor (TLR2) and nuclear factor kappa-B (NF-κB) signaling pathway-related genes were investigated by quantitative real-time PCR. The critical pathway proteins were detected by western blot. The results showed that the multiplicity of infection (MOI; the ratio of bacteria to bMECs) 5:1 of S. haemolyticus for 12 h could cause cellular inflammation, which was selected to establish the inflammatory model. Incubation with 50 µg/ml PF for 12 h was the best intervention condition for cells stimulated by S. hemolyticus. Quantitative real-time PCR and western blot analysis showed that PF inhibited the activation of TLR2 and NF-κB pathway-related genes and the expression of related proteins. Western blot results showed that PF suppressed the expression of NF-κB unit p65, NF-κB unit p50, and MyD88 in bMECs stimulated by S. haemolyticus. The inflammatory response pathway and molecular mechanism caused by S. haemolyticus on bMECs are related to TLR2-mediated NF-κB signaling pathways. The anti-inflammatory mechanism of PF may also be through this pathway. Therefore, PF is expected to develop potential drugs against CoNS-induced bovine mastitis.

Antimicrobial Resistance and Virulence Factor of Streptococcus dysgalactiae Isolated from Clinical Bovine Mastitis Cases in Northwest China.

OBJECTIVE: Streptococcus dysgalactiae is a major pathogen in bovine mastitis. The purpose of this study was to survey the prevalence, antimicrobial resistance, as well as the spread of resistance and virulence-associated gene of S. dysgalactiae. METHODS: A total of 60 S. dysgalactiae strains were obtained from 830 milk samples from Holstein cows with clinical mastitis. Antimicrobial resistance was examined by the disk diffusion method. Antimicrobial resistance and virulence genes were investigated by PCR, agarose gel electrophoresis and 16S rRNA gene sequencing. RESULTS: All isolates were resistant to tetracycline and showed a high level of resistance to aminoglycoside antibiotics, where 81.67% of the strains were multi-resistant to these ten sorts of antibiotics. In addition, the most prevalent resistance gene in S. dysgalactiae was aphA-1 (98.33%), followed by blaTEM (96.67%), ermB (83.3%), aadA1/aadA2 (78.33%) and tetL (73.33%). Totally, seven virulence genes with 25 combination patterns were detected in these isolates, and each isolates harbored at least one virulence gene. 21.67% of the isolates carried three or more virulence genes, while one strain with seven virulence-related genes and belonged to cfb+lmb+eno+napr+bca+scpB+cyl. CONCLUSION: These findings indicate that S. dysgalactiae isolated from clinical bovine mastitis cases in Northwest China show a variety of molecular ecology and are highly resistant to antibiotics commonly used in dairy farms. This research will help investigators better understand the pathophysiology S. dysgalactiae in bovine mastitis and choose the appropriate antibiotics to treat mastitis.