Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

Background

Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.

Methods

Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.

Results

Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.

Conclusion

We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.     

Antibacterial activity and mechanism of chloroform fraction from aqueous extract of mugwort leaves (Artemisia argyi L.) against Staphylococcus aureus

In this work, the antibacterial activity and mechanism of chloroform fraction obtained from aqueous extract of mugwort leaves against Staphylococcus aureus were investigated. The extract showed obvious antibacterial activity against S. aureus which the minimum inhibitory concentration and minimum bactericidal concentration were determined to be 3·0 and 6·0 mg ml⁻¹ respectively. The mechanism study suggested that the extract could destroy the integrity of the S. aureus cell walls and increase the permeability of cell membrane in a certain concentration, but it could not kill S. aureus in a short time. Instead, the extract could make bacteria in a state of apoptosis for a long time, interfere with the normal physiological metabolism of bacteria, and eventually make bacteria die, which was confirm by scanning electronic microscope.     

Inflammatory microenvironment of fibrotic liver promotes hepatocellular carcinoma growth,metastasis and sorafenib resistance through STAT3 activation

The pro-inflammatory and pro-fibrotic liver microenvironment facilitates hepatocarcinogenesis. However, the effects and mechanisms by which the hepatic fibroinflammatory microenvironment modulates intrahepatic hepatocellular carcinoma (HCC) progression and its response to systematic therapy remain largely unexplored. We established a syngeneic orthotopic HCC mouse model with a series of persistent liver injury induced by CCl₄ gavage, which mimic the dynamic effect of hepatic pathology microenvironment on intrahepatic HCC growth and metastasis. Non-invasive bioluminescence imaging was applied to follow tumour progression over time. The effect of the liver microenvironment modulated by hepatic injury on sorafenib resistance was investigated in vivo and in vitro. We found that the persistent liver injury facilitated HCC growth and metastasis, which was positively correlated with the degree of liver inflammation rather than the extent of liver fibrosis. The inflammatory cytokines in liver tissue were clearly increased after liver injury. The two indicated cytokines, tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), both promoted intrahepatic HCC progression via STAT3 activation. In addition, the hepatic inflammatory microenvironment contributed to sorafenib resistance through the anti-apoptotic protein mediated by STAT3, and STAT3 inhibitor S3I-201 significantly improved sorafenib efficacy impaired by liver inflammation. Clinically, the increased inflammation of liver tissues was accompanied with the up-regulated STAT3 activation in HCC. Above all, we concluded that the hepatic inflammatory microenvironment promotes intrahepatic HCC growth, metastasis and sorafenib resistance through activation of STAT3.     

CRISPR/Cas9 knockout of MTA1 enhanced RANKL-induced osteoclastogenesis in RAW264.7 cells partly via increasing ROS activities

Metastasis-associated protein 1 (MTA1), belonging to metastasis-associated proteins (MTA) family, which are integral parts of nucleosome remodelling and histone deacetylation (NuRD) complexes. However, the effect of MTA1 on osteoclastogenesis is unknown. Currently, the regulation of MTA1 in osteoclastogenesis was reported for the first time. MTA1 knockout cells (KO) were established by CRISPR/Cas9 genome editing. RAW264.7 cells with WT and KO group were stimulated independently by RANKL to differentiate into mature osteoclasts. Further, western blotting and quantitative qRT-PCR were used to explore the effect of MTA1 on the expression of osteoclast-associated genes (including CTSK, MMP9, c-Fos and NFATc1) during osteoclastogenesis. Moreover, the effects of MTA1 on the expression of reactive oxygen species (ROS) in osteoclastogenesis was determined by 2′, 7′ -dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Nuclear translocation of Nrf2 was assessed by immunofluorescence staining and western blotting. Our results indicated that the MTA1 deletion group could differentiate into osteoclasts with larger volume and more TRAP positive. In addition, compared with WT group, KO group cells generated more actin rings. Mechanistically, the loss of MTA1 increased the expression of osteoclast-specific markers, including c-Fos, NFATc1, CTSK and MMP-9. Furthermore, the results of qRT-PCR and western blotting showed that MTA1 deficiency reduced basal Nrf2 expression and inhibited Nrf2-mediated expression of related antioxidant enzymes. Immunofluorescence staining demonstrated that MTA1 deficiency inhibited Nrf2 nuclear translocation. Taken together, the above increased basal and RANKL-induced intracellular ROS levels, leading to enhanced osteoclast formation.     

Eight new C-21 steroidal glycosides from Dregea sinensis var. corrugata

Eight new steroidal glycosides, dresiosides were isolated from the stems of Dregea sinensis var. corrugate and their structures were deduced on the basis of chemical and spectral evidence. The sugar units of dresiosides were determined as deoxysugars after hydrolysis and isolation from natural glycosides. The absolute configurations of deoxysugars were determined as D on the basis of their optical rotation values in comparison with appropriate standards.     

Exploration of pairing constraints identifies a 9 base-pair core within box C/D snoRNA-rRNA duplexes

2'-O-ribose methylation of eukaryotic ribosomal RNAs is guided by RNA duplexes consisting of rRNA and box C/D small nucleolar (sno)RNA sequences, the methylated sites invariably mapping five positions apart from the D box. Here we have analyzed the RNA duplex pairing constraints by investigating the features of 415 duplexes from the fungus, plant and animal kingdoms, and the evolution of those duplexes within the 124 sets they group into. The D-box upstream 1st and >or=15th positions consist of Watson-Crick base-pairs, G:U base-pairs and mismatched bases with ratios close to random assortments; these positions display single base differences in >60% of the RNA duplex sets. The D-box upstream 2nd to 11th positions have >90% Watson-Crick base-pairs; they display single base mutations with a U-shaped distribution of lower values of 0% and 1.6% at the methylated site 5th and 4th positions, and double compensatory mutations leading to new Watson-Crick base-pairs with an inverted U-shaped distribution of higher values at the 8th to 11th positions. Half of the single mutations at the 3rd to 11th positions resulted in G:U base-pairing, mainly through A-->G mutations in the rRNA strands and C-->T mutations in the snoRNA strands. Double compensatory mutations at the 3rd to 11th positions are extremely frequent, representing 36% of all mutations; they frequently arose from an A-->G mutation in the rRNA strands followed by a T-->C mutation in the snoRNA strands. Differences in the mutational pathways through which the rRNA and snoRNA strand evolved must be related to differences in the rRNA and snoRNA copy number and gene organization. Altogether these data identify the D-box upstream 3rd to 11th positions as box C/D snoRNA-rRNA duplex cores. The impact of the pairing constraints on the evolution of the 9 base-pair RNA duplex cores is discussed.     

Arylsulfonamides as a new class of cannabinoid CB1 receptor ligands: identification of a lead and initial SAR studies

High-throughput screening of the UCB sample collection identified the piperidinyl-sulfonyl benzoic ester 1 as a novel agonist for CB(1) receptor with nanomolar affinity. We report here the pharmacological profile of compound 1 as well as preliminary biological activities in pain model. Diverse close analogs of 1 were purchased and the structure-affinity relationships among this novel class are discussed.