Differences in Colistin-resistant Acinetobacter baumannii Clinical Isolates Between Patients With and Without Prior Colistin Treatment.

BACKGROUND: The increasing morbidity and mortality rates associated with Acinetobacter baumannii are due to the emergence of drug resistance and the limited treatment options. We compared characteristics of colistin-resistant Acinetobacter baumannii (CR-AB) clinical isolates recovered from patients with and without prior colistin treatment. We assessed whether prior colistin treatment affects the resistance mechanism of CR-AB isolates, mortality rates, and clinical characteristics. Additionally, a proper method for identifying CR-AB was determined. METHODS: We collected 36 non-duplicate CR-AB clinical isolates resistant to colistin. Antimicrobial susceptibility testing, Sanger sequencing analysis, molecular typing, lipid A structure analysis, and in vitro synergy testing were performed. Eleven colistin-susceptible AB isolates were used as controls. RESULTS: Despite no differences in clinical characteristics between patients with and without prior colistin treatment, resistance-causing genetic mutations were more frequent in isolates from colistin-treated patients. Distinct mutations were overlooked via the Sanger sequencing method, perhaps because of a masking effect by the colistin-susceptible AB subpopulation of CR-AB isolates lacking genetic mutations. However, modified lipid A analysis revealed colistin resistance peaks, despite the population heterogeneity, and peak levels were significantly different between the groups. CONCLUSIONS: Although prior colistin use did not induce clinical or susceptibility differences, we demonstrated that identification of CR-AB by sequencing is insufficient. We propose that population heterogeneity has a masking effect, especially in colistin non-treated patients; therefore, accurate testing methods reflecting physiological alterations of the bacteria, such as phosphoethanolamine-modified lipid A identification by matrix-assisted laser desorption ionization-time of flight, should be employed.

Lipid A-based affinity biosensor for screening anti-sepsis components from herbs.

LPS (lipopolysaccharide), an outer membrane component of Gram-negative bacteria, plays an important role in the pathogenesis of sepsis and lipid A is known to be essential for its toxicity. Therefore it could be an effective measure to prevent sepsis by neutralizing or destroying LPS. Numerous studies have indicated that many traditional Chinese medicines are natural antagonists of LPS in vitro and in vivo. The goal of this study is to develop a rapid method to screen anti-sepsis components from Chinese herbs by use of a direct lipid A-based affinity biosensor technology based on a resonant mirror. The detergent OG (n-octyl ß-D-glucopyranoside) was immobilized on a planar non-derivatized cuvette which provided an alternative surface to bind the terminal hydrophilic group of lipid A. A total of 78 herbs were screened based on the affinity biosensor with a target of lipid A. The aqueous extract of PSA (Paeonia suffruticosa Andr) was found to possess the highest capability of binding lipid A. Therefore an aqueous extraction from this plant was investigated further by our affinity biosensor, polyamide chromatography and IEC-HPLC. Finally, we obtained a component (PSA-I-3) from Paeonia suffruticosa Andr that was evaluated with the affinity biosensor. We also studied the biological activities of PSA-I-3 against sepsis in vitro and in vivo to further confirm the component we screened with the biosensor. In vitro, we found that PSA-I-3 could decrease TNFα (tumour necrosis factor α) release from RAW264.7 cells induced by LPS in a dose-dependent manner. In vivo, it increased remarkably the survival of KM (KunMing) mice by challenging both lethal-dose LPS and heat-killed Escherichia coli compared with control groups. Our results suggest that the constructed affinity biosensor can successfully screen the anti-sepsis component from Chinese herbs.

Improvement for high fat diet-induced hepatic injuries and oxidative stress by flavonoid-enriched extract from Nelumbo nucifera leaf.

Nelumbo nucifera Gaertn is widespread and a popular food in central and southern Taiwan. It has also been reported to possess different therapeutic effects, but the effects of N. nucifera leaf on lipid metabolism and liver function remain unclear. In this study, a high fat diet was used to induce hyperlipidemia, hypercholesterolemia, and fatty liver in hamster. The effects of flavonoid-enriched N. nucifera leaf extract supplement and two lipid-lowing drugs, silymarin and simvastatin, on the disorders induced by high fat diet were investigated. The results showed that a 10-week application of a high fat diet to hamsters led to significant increases of body weight, plasma lipid derivatives (triglyceride, total cholesterol, and lipoproteins), lipid peroxidation, and liver damage markers (plasma aspartate aminotransferase and alanine aminotransferase). Interestingly, flavonoid-enriched N. nucifera leaf extract supplement effectively ameliorated the high fat diet-induced lipid metabolic disorders as significantly as silymarin and simvastatin did. Moreover, the flavonoid-enriched supplement alleviated the high fat diet-induced accumulation of lipids in liver, the findings showing distinguishing mechanisms from the effects of silymarin and simvastatin. These results suggested that the flavonoid-enriched N. nucifera leaf extract supplement may significantly improve the high fat diet-induced abnormal blood lipids and liver damage as significantly as the common drugs. Consequently, it is suggested that the flavonoid-enriched N. nucifera leaf extract supplement is beneficial for the improvement of lipid metabolisms and the alleviation of liver damage in high fat diet treatment.

Effects of dehulled adlay on plasma glucose and lipid concentrations in streptozotocin-induced diabetic rats fed a diet enriched in cholesterol.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) is a cereal food for humans and has been also used as a superior medical herb substance and functional food for traditional treatment of diabetes in China. However, its scientific basis as a functional food is still unclear. The purpose of this study was to investigate the effect of dietary dehulled adlay on plasma lipid and glucose concentrations in diabetic rats. The diabetic male Sprague-Dawley (SD) rats, induced by injection of streptozotocin (60 mg/kg subcutaneously), were fed a cholesterol-rich diet (0.5% cholesterol) containing corn starch or dehulled adlay for four weeks. After completion of the experimental period, the abdominal adipose tissue and liver of rats were excised and weighed, and the plasma glucose, triglyceride, and lipoprotein cholesterol concentrations were assayed. The results showed that diabetic rats fed a dehulled adlay diet exhibited a greater adipose tissue weight (9.36 +/- 3.43 vs. 5.39 +/- 3.04 g, p < 0.05) and a reduced food intake (39.3 +/- 5.9 vs. 61.0 +/- 11.7 g/day, p < 0.05) when compared with animals fed a cornstarch diet. Significantly decreased plasma glucose (261.6 +/- 96.6 vs. 422.1 +/- 125.4 mg/dL, p < 0.05), total cholesterol (289.4 +/- 140.6 vs. 627.3 +/- 230.5 mg/dL, p < 0.05), and triglyceride (52.3 +/- 14.4 vs. 96.5 +/- 36.6 mg/dL, p < 0.05) levels were observed in rats fed the dehulled adlay diet. In addition, the ingestion of dehulled adlay appears to significantly decrease plasma low-density lipoprotein (LDL) plus very low-density lipoprotein (VLDL) cholesterol concentrations. Rats fed a dehulled adlay diet showed an increase in fecal weight and cholesterol contents of stools. Although a significantly decreased plasma thiobarbituric reactive substances (TBARS) value was observed in diabetic rats fed the dehulled adlay diet (6.2 +/- 3.4 vs. 11.0 +/- 3.8 nmol malondialdehyde (MDA)/mL, p < 0.05), no significant difference in the hepatic TBARS value was observed between the two dietary groups. Results from the present study suggest that dehulled adlay exhibited not only a hypolipidemic effect but also displayed a hypoglycemic ability in diabetic rats, indicating that dehulled adlay may play an important role in the regulation of plasma lipid and glucose metabolisms in diabetic rats induced by streptozotocin.

Antigenic and phenotypic modifications of Yersinia pestis under calcium and glucose concentrations simulating the mammalian bloodstream environment.

To study the possible mechanism of extracellular resistance to phagocytes developed by Yersinia pestis in the early stage of plague infection, the behaviour of two Y. pestis strains, the vaccine EV-76 and fully virulent 231 (LD(50), 10 c.f.u.), was studied in-depth after cultivation in vitro at the host temperature in conditions simulating the bloodstream environment of mammals. For this, two standard basal media supplemented with calcium and glucose in appropriate concentrations were employed: Hottinger broth, routinely used for growth of Y. pestis in vitro, and RPMI 1640, simulating human extracellular fluid. Although both media permitted Y. pestis to achieve the resistant state, RPMI enabled significantly higher bacterial proliferation and increased modifications in the production of the principal surface antigens that affect the relevant phenotype characteristics. In general, our results indicate that the Y. pestis bacteria in the resistant state do not produce species-specific antigens, i.e. fraction 1 or F1, 'murine' toxin or Ymt, plasminogen activator (Pla) and any surface-specific polysaccharides, resulting in unmasking of the cross-reactive epitopes of lipid A in reduced Y. pestis lipopolysaccharide. This may produce mimicry by Y. pestis of some human tissue and blood cell components, with no immune response and inflammation at the site of infection at the early stage, which enables Y. pestis to survive, extensively multiply and spread into the circulation.

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus.

Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an O-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an O-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.

Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Location of acyl groups at the lipid A backbone.

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12 degrees C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetra-decanoic acid at GlcN II is positionally replaced by delta 9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to GlcN II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to GlcN I are free or only partially substituted.

[Chemotaxonomic characteristics of bacteria belonging to the genus Nocardia isolated from Ukrainian SSR soils]. / Khemotaksonomicheskie priznaki bakterii roda Nocardia, izolirovannykh iz pochv USSR

From soils of the Ukrainian SSR bearing oil and without it, 241 strains of Nocardia were isolated on mineral media containing n-alkanes C12-C22. The strains were divided into two groups: actinoid (N. asteroides, N. flavescens) and bacterium-like (N. rubropertincta, N. flava, N. ucrainica, N. salmonicolor, N. rubra, N. erythropolis, Nocardia ssp). Chemotaxonomic characteristics (the presence in the cells of galactose, arabinose, mesodiaminopimelic acid, lipid LCN-A) were used to differentiate Nocardia from other genera of actinomycetes and coryneforms and to confirm the taxonomy of museum strains. The majority of the Nocardia cultures belongs to the groups "asteroides" and "rhodochrous" according to location of spots of lipid LCN-A on chromatograms. The strains of N. asteroides and complex "M." rhodochrous may belong to both groups. A small number of strains belongs to the group which is represented by N. calcarea 41 (NCIB 8863).

Biological activities of endotoxins from Yersinia enterocolitica.

The chemical properties and the general biological activities of lipopolysaccharide (LPS) and Boivin-type endotoxin obtained respectively by phenol-water and trichloroacetic acid extraction from Yersinia enterocolitica serotypes O3 and O9 were studied. The yield of LPS from the O9 strain was about 10% of the O3 strain possibly because of the lower solubility of O9-LPS in aqueous phase. However, the chemical composition of O9-LPS was similar to that of O3-LPS in the proportions of reducing sugar, glucosamine, heptose, KDO, and lipid A. In pyrogenicity and local Shwartzman reactivity in rabbits and lethality for mice, there was also no difference between O3 and O9-LPS. The anthrone-positive carbohydrate and lipid A contents of Boivin-type endotoxin from O3 were higher than those of the endotoxin from O9. The biological activities of Boivin-type endotoxin from O3 were also remarkably higher than those of the endotoxin from O9. It seems that endotoxin of Y. enterocolitica serotype O3 may play an important role in infection by this organism.