Staphylococcus epidermidis is a leading cause of hospital-acquired infections, mostly associated with the use of medical devices in seriously ill or immunocompromised patients. Currently, the clonal characteristics of S. epidermidis in the hospital environment in China are unknown; neither is it known why these sequence types are easily disseminated in the hospital setting. In this study, multilocus sequence typing (MLST) was employed for the clonal analysis of 80 S. epidermidis isolates collected from patients with S. epidermidis infections. MLST revealed a total of 16 different sequence types among these isolates. ST2, which contained exclusively ica-positive, IS256-positive and biofilm-forming isolates, represented the majority of clinical strains tested. Of the S. epidermidis strains circulating in the hospital environment in China, as many as 96.25 % are resistant to meticillin. Four staphylococcal chromosomal cassette mec (SCCmec) types were identified among the total 80 S. epidermidis isolates, none of the strains carried an SCCmec I cassette. All of the ST2 isolates carried the SCCmec type III cassette. Taken together, the combination of biofilm-forming ability and antibiotic resistance helps ST2 become successfully established within nosocomial environments, and promotes the device-related infection and bacteraemia. INTRODUCTIONStaphylococci are one of the most important causes of nosocomial infections, from minor skin infections to lifethreatening bacteraemia (Monk et al., 2008;Francois et al., 2008; Miragaia et al., 2008). The two major opportunistic pathogens of this genus, Staphylococcus aureus and Staphylococcus epidermidis, colonize a sizeable proportion of the human population. Because S. aureus can produce a greater variety of exotoxins and enterotoxins compared to S. epidermidis it is a more aggressive pathogen, in recent years, a lot of work has been focused on this bacteria Otto, 2008). Compared to S. aureus, there is a scarcity of information concerning the molecular characteristics of S. epidermidis, especially in the hospital setting in China.Biofilm formation is the most important factor for the establishment of S. epidermidis as a nosocomial pathogen (Hansen et al., 2007). Although the production of staphylococcal biofilm is dependent upon multiple regulatory proteins, an essential factor is the presence and expression of the four gene icaADBC operon (Li et al., 2005). It was reported recently that an insertion sequence element called IS256 has the capacity to influence expression of the ica operon, and subsequent biofilm formation, by reversible insertion into the ica operon and its regulatory genes, as well as by chromosomal rearrangement (Valle et al., 2007). S. epidermidis is a polymorphic species that was found to have a clonal population structure. With respect to the ica and IS256 operon, it is not currently known from where this genetic information originates, and there is uncertainty as to whether biofilmforming strains disseminate clonally or by horizontal transfer of the ica a...

New therapeutic approaches are urgently needed to improve survival outcomes for patients with necrotizing pneumonia caused by Staphylococcus aureus One such approach is adjunctive treatment with intravenous immunoglobulin (IVIG), but clinical practice guidelines offer conflicting recommendations. In a preclinical rabbit model, prophylaxis with IVIG conferred protection against necrotizing pneumonia caused by five different epidemic strains of community-associated methicillin-resistant S. aureus (MRSA) as well as a widespread strain of hospital-associated MRSA. Treatment with IVIG, either alone or in combination with vancomycin or linezolid, improved survival outcomes in this rabbit model. Two specific IVIG antibodies that neutralized the toxic effects of α-hemolysin (Hla) and Panton-Valentine leukocidin (PVL) conferred protection against necrotizing pneumonia in the rabbit model. This mechanism of action of IVIG was uncovered by analyzing loss-of-function mutant bacterial strains containing deletions in 17 genes encoding staphylococcal exotoxins, which revealed only Hla and PVL as having an impact on necrotizing pneumonia. These results demonstrate the potential clinical utility of IVIG in the treatment of severe pneumonia induced by S. aureus.

Genes all5292 (cpcS2) and alr0617 (cpcS1) in the cyanobacterium Nostoc PCC7120 are homologous to the biliprotein lyase cpcS, and genes all5339 (cpcT1) and alr0647 (cpcT2) are homologous to the lyase cpcT. The functions of the encoded proteins were screened in vitro and in a heterologous Escherichia coli system with plasmids conferring biosynthesis of the phycocyanobilin chromophore and of the acceptor proteins ␤-phycoerythrocyanin (PecB) or ␤-phycocyanin (CpcB). CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine ␤155 but does not discriminate between CpcB and PecB. The in vitro reconstitutions required no cofactors, and kinetic constants were determined for CpcT1 under in vitro conditions. No lyase activity was found for the lyase homologues CpcS2 and CpcT2, but complexes are formed in vitro between CpcT1 and CpcS1, CpcT2, or PecE (subunit of phycoviolobilin:␣-phycoerythrocyanin isomerase lyase). The genes coding the inactive homologues, cpcS2 and cpcT2, are transcribed in N-starved Nostoc. In sequential binding experiments with CpcT1 and CpcS1, a chromophore at cysteine 84 inhibited the subsequent attachment to cysteine 155, whereas the inverse sequence generates subunits carrying both chromophores.

Summary Celastrol is a promising bioactive compound isolated from Tripterygium wilfordii and has been shown to possess many encouraging preclinical applications. However, the celastrol biosynthetic pathway is poorly understood, especially the key oxidosqualene cyclase (OSC) enzyme responsible for cyclisation of the main scaffold. Here, we report on the isolation and characterisation of three OSCs from T. wilfordii: TwOSC1, TwOSC2 and TwOSC3. Both TwOSC1 and TwOSC3 were multiproduct friedelin synthases, while TwOSC2 was a β‐amyrin synthase. We further found that TwOSC1 and TwOSC3 were involved in the biosynthesis of celastrol and that their common product, friedelin, was a precursor of celastrol. We then reconstituted the biosynthetic pathway of friedelin in engineered yeast constructed by the CRISPR/Cas9 system, with protein modification and medium optimisation, leading to heterologous production of friedelin at 37.07 mg l−1 in a shake flask culture. Our study was the first to identify the genes responsible for biosynthesis of the main scaffold of celastrol and other triterpenes in T. wilfordii. As friedelin has been found in many plants, the results and approaches described here have laid a solid foundation for further explaining the biosynthesis of celastrol and related triterpenoids. Moreover, our results provide insights for metabolic engineering of friedelane‐type triterpenes.

Staphylococcus aureus is a globally important human pathogen, especially among children and immunocompromised patients. The emergence and spread of community-associated methicillin-resistant S. aureus (CA-MRSA) has become a serious public health problem worldwide. The aim of this study was to investigate the prevalence, molecular characteristics and virulence profiles of CA-MRSA infections from pediatric patients in a university hospital in Shanghai, China. A total of 80 CA-MRSA isolates were collected from July 2012 to December 2013 in Shanghai Children's Medical Center and analyzed by multilocus sequence typing, staphylococcus chromosomal cassette mec (SCCmec) typing, and spa typing. The detection of Panton-Valentine Leukocidin (pvl), superantigenic and exfoliative toxins, and adhesin genes was also performed. Overall, 16 distinct sequence types (STs) were identified among the 80 isolates. Among them, ST59 was found to be the most prevalent, followed by ST398 (11.3%, 9/80) and ST88 (8.8%, 7/80). SCCmec types IV and V were observed, at 60 and 40%, respectively. Thirty spa types were identified, spa t437 (23.8%) was the most predominant type. All 80 isolates exhibited carriage of at least four virulence genes. Thirty-four (42.5%, 34/80) isolates harbored ≥10 tested virulence genes. Adhesion genes were present in most of the MRSA isolates, including the following: icaA (100%), clfA (100%), sdrC (95%), and sdrE (63.8%). The prevalence of pvl gene was 20%, and multidrug resistance was observed in 36% of all strains. In addition, ST59-MRSA-IV with t437 accounted for 21.3% of occurrences, making it the most prevalent clone. Isolates that were carriers of toxin genes, and hla (100%) and hlg (87.5%) were the most frequent. In conclusion, simultaneous carriage of multiple virulence genes and genetically considerable diversity were very common among CA-MRSA from pediatric patients in Shanghai. ST59-MRSA-IV with t437 was still the most predominant type. The combination of virulence gene profiles and antibiotic resistance may help ST59 to be successfully spread among children.