Effects of subinhibitory concentrations of chlorhexidine and mupirocin on biofilm formation in clinical meticillin-resistant Staphylococcus aureus.

BACKGROUND: The effects of subinhibitory concentrations (sub-MICs) of antibacterial agents on the biofilm-forming ability of Staphylococcus aureus require further study. AIM: To investigate the effects of sub-MICs of chlorhexidine and mupirocin on biofilm formation in clinical meticillin-resistant Staphylococcus aureus (MRSA) isolates. METHODS: MRSA isolates were collected from patients with bloodstream infections at a tertiary care hospital. The basal level of biofilm formation and biofilm induction by sub-MICs of chlorhexidine and mupirocin were evaluated by measuring biofilm mass stained with Crystal Violet. FINDINGS: Of the 112 MRSA isolates tested, 63 (56.3%) and 44 (39.3%) belonged to sequence type (ST)5 and ST72 lineages, respectively, which are the predominant healthcare- and community-associated clones in South Korea. ST5 isolates were more likely to have chlorhexidine MIC ≥4 (73.0% vs 29.5%), resistance to mupirocin (23.8% vs 0%), agr dysfunction (73.0% vs 9.1%), and qacA/B gene (58.7% vs 2.3%) compared to ST72 isolates. The basal level of biofilm formation ability was frequently stronger in ST72 isolates compared to ST5 isolates (77.3% vs 12.7%). Sub-MICs of chlorhexidine and mupirocin promoted biofilm formation in 56.3% and 53.6%, respectively, of all isolates. Biofilm induction was more prevalent in ST5 isolates (85.7% for chlorhexidine, 69.8% for mupirocin) than in ST72 isolates (15.9% for chlorhexidine, 27.3% for mupirocin). CONCLUSION: Sub-MICs of chlorhexidine and mupirocin promoted biofilm formation in half of the clinical MRSA isolates. Our results suggest that ST5 MRSA biofilm can be induced together with some other bacterial virulent factors following exposure to chlorhexidine, which might confer a survival advantage to this clone in the healthcare environment.

Extensive severe fever with thrombocytopenia syndrome virus contamination in surrounding environment in patient rooms.

OBJECTIVES: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease in Korea and China. Although there is previous evidence of person-to-person transmission via direct contact with body fluids, the role of environmental contamination by SFTS virus (SFTSV) in healthcare settings has not been established. We therefore investigated the contamination of the healthcare environment by SFTSV. METHODS: We investigated the possible contamination of hospital air and surfaces with SFTSV transmission by collecting air and swabbing environmental surface samples in two hospitals treating six SFTS patients between March and September 2017. The samples were tested using real-time RT-PCR for SFTS M and S segments. RESULTS: Of the six SFTS patients, four received mechanical ventilation and three died. Five rooms were occupied by those using mechanical ventilation or total plasma exchange therapy in isolation rooms without negative pressure and one room was occupied by a patient bedridden due to SFTS. SFTSV was detected in 14 (21%) of 67 swab samples. Five of 24 swab samples were obtained from fomites including stethoscopes, and 9 of 43 were obtained from fixed structures including doorknobs and bed guardrails. Some samples from fixed structures such as television monitors and sink tables were obtained in areas remote from the patients. SFTSV RNA was not detected in five air samples from three patients' rooms. CONCLUSIONS: Our data suggest that SFTSV contamination was extensive in surrounding environments in SFTS patients' rooms. Therefore, more strict isolation methods and disinfecting procedures should be considered when managing SFTS patients.

Association of negatively-charged phospholipids with low-density lipoprotein (LDL) increases its uptake and the deposition of cholesteryl esters by macrophages.

LDL, the major carrier of cholesterol in blood, is poorly metabolized by macrophages. In contrast, macrophages can recognize and endocytose anionic phospholipids such as phosphatidylserine, phosphatidylglycerol and cardiolipin. Since macrophages can take up large amounts of these phospholipids, experiments were performed to ascertain whether pre-incubation of native LDL with negatively-charged phospholipids would enhance the metabolism of LDL by macrophages. When 125I-LDL was incubated with cardiolipin liposomes for 18 h at 37 degrees C before addition to macrophages, an approx. 40-fold increase of LDL metabolism by these cells was observed. Similar results were found when LDL was pre-incubated with phosphatidylserine or phosphatidylglycerol; however, pre-incubation of LDL with phosphatidylcholine liposomes did not lead to an increase of LDL metabolism. The macrophage uptake of LDL pre-incubated with cardiolipin was reduced to approx. 40% of control values in the presence of dextran sulfate and fucoidin, inhibitors of anionic phospholipid uptake. Cytochalasin D, an inhibitor of phagocytosis, reduced the lysosomal degradation of LDL pre-incubated with cardiolipin to approx. 10% of control values. When the LDL-cardiolipin mixture was chromatographed on agarose gel, two peaks containing LDL were observed in the elution profile: the first peak appeared at the void volume and the second peak was detected just ahead of native LDL. The LDL in both peaks was much more extensively metabolized by macrophages than was native LDL; the LDL in the first peak was metabolized at a rate that was 8 times the second peak. The results demonstrate that negatively-charged phospholipids can form a complex with LDL which facilitates its phagocytosis by macrophages.

Iron-ascorbate-phospholipid mediated modification of low density lipoprotein.

LDL can be oxidized by a variety of agents to form a modified lipoprotein which is capable of being avidly metabolized by macrophages. While previous in vitro studies have focused exclusively on the oxidation of LDL, other lipids found in the atheroma are also subject to oxidation and its lipoperoxide byproducts may contribute to the process of LDL modification. To examine the relationship between the oxidation of phospholipids and the subsequent modification of LDL, we incubated 250 microM phosphatidylcholine with 10 microM ferrous sulfate and 50 microM ascorbic acid in 10 mM Tris (pH 7.0). After 18 h at 37 degrees C, significant amounts of thiobarbituric acid reactive substances (TBARS) were formed. The inclusion of LDL (100 micrograms protein/ml) elevated the TBARS and increased the electrophoretic mobility of the lipoprotein. LDL treated with iron and ascorbate in the absence of phosphatidylcholine did not result in the modification of this lipoprotein. LDL that was incubated with phosphatidylcholine, iron and ascorbate was found to be metabolized by macrophages to a far greater extent than native LDL or LDL treated with phosphatidylcholine alone. Probucol (10 microM) inhibited the LDL modification process. These results demonstrate that while iron and ascorbate cannot oxidize LDL directly, the addition of phosphatidylcholine to these initiators of lipid peroxidation can mediate and lead to the modification of LDL.

Cholesteryl esterase-treated LDL augments oxidized LDL-mediated cholesteryl ester deposition in mouse peritoneal macrophages.

Arterial unesterified cholesterol, phospholipid particles have been isolated from atherosclerotic lesions and characterized. However, the role of these 'liposomes' in macrophage foam cell formation is unclear. Recently, LDL, after trypsin and cholesteryl esterase treatment (T/CE LDL), was shown to have physical properties similar to the unesterified cholesterol, phospholipid particles isolated from atherosclerotic lesions. Yet, when mouse peritoneal macrophages were incubated with these model particles in culture medium (DMEM and 5% LPDS), only an insignificant accumulation of cellular cholesteryl esters was observed. Previously, we demonstrated that complex formation between unesterified cholesterol, phosphatidylcholine liposomes and cupric sulfate-oxidized LDL dramatically enhances the ability of the liposomes to augment cellular cholesterol accretion (Greenspan P, Yu H, Mao F, Gutman RL. J Lipid Res 1997;38:101-109). When T/CE LDL, another cholesterol-rich phospholipid particle, was substituted for unesterified cholesterol phosphatidylcholine liposomes in our complex, mouse peritoneal macrophages accumulated a significant amount of both cellular unesterifed cholesterol (61 microg/mg cell protein) and cholesteryl esters (76 microg/mg cell protein) after 48 h of incubation. These results demonstrate again that the interaction of two cholesterol-bearing particles (T/CE LDL and oxidized LDL), which individually can not promote significant cholesterol accumulation in cells, will, when combined, produce macrophage foam cells.

Advances in agarose gel electrophoresis of serum lipoproteins.

Agarose gel electrophoresis has been extensively employed by researchers to gain a greater understanding of lipoprotein biology and its relationship to cardiovascular disease. Advances in this technique have been made in the visualization and quantitation of separated lipoproteins, in the use of agarose gel electrophoresis for detection and quantitation of apolipoproteins of the separated lipoproteins, and in the detection of lipoprotein heterogeneity. Agarose gel electrophoresis has been employed for two-dimensional electrophoretic analysis of lipoproteins as well as in several different methods which probe the immunological properties of lipoproteins. Agarose gel electrophoresis has thus become an important tool in the study of serum lipoproteins in both clinical and basic science laboratories.

A new DNA-dependent ATPase which stimulates yeast DNA polymerase I and has DNA-unwinding activity.

Two forms of DNA-dependent ATPase activity were previously purified from the yeast Saccharomyces cerevisiae and characterized (Plevani, P., Badaracco, G., and Chang, L. M. S. (1980) J. Biol. Chem. 255, 4957-4963). Here, an additional DNA-dependent ATPase (ATPase III) has been purified from S. cerevisiae to near homogeneity. This ATPase differs from those described previously in its chromatographic properties, molecular weight, reaction properties and immunological relatedness. Its molecular weight is about 63,000 in the presence of sodium dodecyl sulfate. It hydrolyzes ATP to ADP and orthophosphate in the presence of DNA as an effector. In addition, yeast DNA polymerase I, which is a true DNA replicase of yeast, is stimulated severalfold by this ATPase. Neither yeast DNA polymerase II nor prokaryotic DNA polymerases are stimulated. This stimulation is intrinsic to the ATPase activity, since both activities copurified in the last four steps of purification, showed the same heat stability and showed dependence on and hydrolysis of ATP. The ATPase III preparation also contains a DNA-unwinding (DNA helicase) activity, which unwinds double-stranded DNA in the presence of ATP. In the S. cerevisiae radiation-sensitive mutant rad3, no significant ATPase III activity could be detected, suggesting that the RAD3 gene, which codes for a different polypeptide, regulates the expression of ATPase III activity.

Cholesteryl ester accumulation in macrophages treated with oxidized low density lipoprotein.

The ability of CuSO4- and hypochlorite-oxidized LDL to promote cholesterol accumulation in macrophages was examined. Both CuSO4- and hypochlorite-oxidized LDL were rapidly metabolized by mouse peritoneal macrophages to a level approximately 10 times that observed for native LDL and both modified lipoproteins increased the accumulation of unesterified cholesterol. However when each modified lipoprotein was incubated with macrophages for 40h, only hypochlorite-oxidized LDL produced significant accumulation of cholesteryl esters, with levels approaching 85 micrograms/mg cell protein. This finding was verified by nile red staining. The cholesteryl ester content of cupric sulfate-modified LDL was found to be significantly decreased when compared to either native or hypochlorite-modified LDL promotes massive cholesteryl ester accumulation because the cholesteryl ester content of the LDL particle is preserved.