Relationship between L-lactate dehydrogenase and multidrug resistance in Staphylococcus xylosus.

Staphylococcus xylosus is a gram-positive bacterium that has attracted much attention due to its increasing clinical appearance, frequently associated with serious multidrug resistance cases. L-lactate dehydrogenase (LDH) has been related to drug resistance in several bacterial species. However, the mechanism of multidrug resistance in S. xylosus remains unclear as well as the involvement of LDH in such resistance. To explore the relationship between multidrug resistance and LDH in S. xylosus, we used tylosin-resistant S. xylosus as the parent strain to construct ldh knockout and complemented strains. Then, we tested their resistance to macrolides, lincosamides, tetracyclines, and aminoglycosides. In addition, the enzyme activity, metabolite content, and transcriptional level of key genes involved in the TCA cycle and thioredoxin system were determined to clarify the mechanism of resistance. We observed that the resistance to multiple antibiotics increased significantly after ldh knockout, especially that to lincomycin, whereas antibiotic sensitivity was partially restored in the complemented strain. The levels of pyruvate, nicotinamide adenine dinucleotide, and reactive oxygen species decreased significantly upon ldh knockout, and the activity of isocitrate dehydrogenase and malate dehydrogenase decreased. These results indicate that the lack of LDH promotes multidrug resistance in S. xylosus by inhibiting the TCA cycle and regulating the thioredoxin system.

Albendazole solid dispersions prepared using PEG6000 and Poloxamer188: formulation, characterization and in vivo evaluation.

This study aimed to optimize the preparation process of albendazole (ABZ) solid dispersion (SD) and enhance its dissolution rate and oral bioavailability in dogs. The ABZ-SD formulations were prepared by a fusion method with ABZ and polyethylene glycol 6000 (PEG 6000), poloxamer 188 (P 188) polymers at various weight ratios or the combination of PEG 6000&P 188. The characterizations of the optimal formulations were performed by scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), in vitro dissolution test and molecular docking. The in vivo pharmacokinetic study was conducted in beagle dogs. As a result, ABZ solid dispersion based on PEG 6000&P 188 (1:2) was successfully prepared. The ABZ-SD formulation could significantly improve the apparent solubility and dissolution rate of ABZ compared with commercial tablets. Furthermore, the water solubility of ABZ-SD was improved mainly based on hydrogen bond association. Besides, at an oral dosage of 15 mg/kg ABZ, the SDs had higher Cmax values and areas under the curve (AUCs) compared to those of commercial ABZ tablets. Preparation of ABZ-loaded SDs by PEG 6000&P 188 is a promising strategy to improve the oral bioavailability of ABZ.

The Active Ingredients Identification and Antidiarrheal Mechanism Analysis of Plantago asiatica L. Superfine Powder.

Plantago asiatica L. is a natural medicinal plant that has been widely used for its various pharmacological effects such as antidiarrheal, anti-inflammatory, and wound healing. This study aims to explore the antidiarrheal active ingredients of Plantago asiatica L. that can be used as quality markers to evaluate P. asiatica L. superfine powder (PSP). Molecular docking experiment was performed to identify the effective components of P. asiatica L., which were further evaluated by an established mouse diarrhea model. Na+/K+-ATPase and creatine kinase (CK) activities and the Na+/K+ concentrations were determined. The gene expression of ckb and Atp1b3 was detected. PSP was prepared and evaluated in terms of the tap density and the angle of repose. The structures of PSPs of different sizes were measured by infrared spectra. The active ingredient contents of PSPs were determined by HPLC. The results indicated that the main antidiarrheal components of P. asiatica L. were luteolin and scutellarein that could increase the concentration of Na+ and K+ by upregulating the activity and gene level of CK and Na+/K+-ATPase. In addition, luteolin and scutellarein could also decrease the volume and weight of small intestinal contents to exert antidiarrheal activity. Moreover, as the PSP size decreased from 6.66 to 3.55 µm, the powder tended to be amorphous and homogenized and of good fluidity, the content of active compounds gradually increased, and the main structure of the molecule remained steady. The optimum particle size of PSP with the highest content of active components was 3.55 µm, and the lowest effective dose for antidiarrhea was 2,000 mg/kg. Therefore, the antidiarrheal active ingredients of PSP were identified as luteolin and scutellarein that exert antidiarrheal activity by binding with Na+/K+-ATPase. PSP was successfully prepared and could be used as a new dosage form for the diarrhea treatment.

Analysis of multidrug resistance in Streptococcus suis ATCC 700794 under tylosin stress.

Streptococcus suis is an important zoonotic pathogen. The massive use of tylosin and other antibiotics in swine production has led to the emergence of resistant phenotypes of S. suis. However, there are no adequate measures available to address the problem of bacterial resistance. This study involved the use of 1/4 MIC (0.125 µg/mL) of tylosin to investigate resistance-related proteins by S. suis ATCC 700794. Our results showed that 171 proteins were differentially expressed in S. suis tested with 1/4 MIC (0.125 µg/mL) of tylosin using iTRAQ-based quantitative proteomic methods. TCS, heat shock protein and elongation factors were differentially expressed at 1/4 MIC (0.125 µg/mL) of tylosin compared to non treated, control cells. Using quantitative RT-PCR analysis, we verified the relationship between the differentially expressed proteins in S. suis with different MIC values. The data showed that expression profile for elongation factor G (fusA), elongation factor Ts (tsf), elongation factor Tu (tuf), putative histidine kinase of the competence regulon, ComD (comD), putative competence-damage inducible protein (cinA) and protein GrpE (grpE), observed in tylosin-resistant S. suis, correlated with that of S. suis ATCC 700794 at 1/4 MIC (0.125 µg/mL). The MIC of tylosin-resistant showed high-level resistance in terramycin, chlortetracycline, ofloxacin and enrofloxacin. Our findings demonstrated the importance of elongation factors, TCS and heat shock protein during development of tylosin resistance in S. suis. Thus, our study will provide insight into new drug targets and help reduce bacterial multidrug resistance through development of corresponding inhibitors.

Transcriptomic analysis reveals flavonoid biosynthesis of Syringa oblata Lindl. in response to different light intensity.

BACKGROUND: Hazy weather significantly increase air pollution and affect light intensity which may also affect medicinal plants growth. Syringa oblata Lindl. (S. oblata), an effective anti-biofilm medicinal plants, is also vulnerable to changes in plant photoperiods and other abiotic stress responses. Rutin, one of the flavonoids, is the main bioactive ingredient in S. oblata that inhibits Streptococcus suis biofilm formation. Thus, the present study aims to explore the biosynthesis and molecular basis of flavonoids in S. oblata in response to different light intensity. RESULTS: In this study, it was shown that compared with natural (Z0) and 25% ~ 35% (Z2) light intensities, the rutin content of S. oblata under 50% ~ 60% (Z1) light intensity increased significantly. In addition, an integrated analysis of metabolome and transcriptome was performed using light intensity stress conditions from two kinds of light intensities which S. oblata was subjected to: Z0 and Z1. The results revealed that differential metabolites and genes were mainly related to the flavonoid biosynthetic pathway. We found out that 13 putative structural genes and a transcription factor bHLH were significantly up-regulated in Z1. Among them, integration analysis showed that 3 putative structural genes including 4CL1, CYP73A and CYP75B1 significantly up-regulated the rutin biosynthesis, suggesting that these putative genes may be involved in regulating the flavonoid biosynthetic pathway, thereby making them key target genes in the whole metabolic process. CONCLUSIONS: The present study provided helpful information to search for the novel putative genes that are potential targets for S. oblata in response to light intensity.

Comparative proteomic analysis reveals drug resistance of Staphylococcus xylosus ATCC700404 under tylosin stress.

BACKGROUND: As a kind of opportunist pathogen, Staphylococcus xylosus (S. xylosus) can cause mastitis. Antibiotics are widely used for treating infected animals and tylosin is a member of such group. Thus, the continuous use of antibiotics in dairy livestock enterprise will go a long way in increasing tylosin resistance. However, the mechanism of tylosin-resistant S. xylosus is not clear. Here, isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods was used to find resistance-related proteins. RESULTS: We compared the differential expression of S. xylosus in response to tylosin stress by iTRAQ. A total of 155 proteins (59 up-regulated, 96 down-regulated) with the fold-change of >1.2 or <0.8 (p value ≤0.05) were observed between the S. xylosus treated with 1/2 MIC (0.25 µg/mL) tylosin and the untreated S. xylosus. Bioinformatic analysis revealed that these proteins play important roles in stress-response and transcription. Then, in order to verify the relationship between the above changed proteins and mechanism of tylosin-resistant S. xylosus, we induced the tylosin-resistant S. xylosus, and performed quantitative PCR analysis to verify the changes in the transcription proteins and the stress-response proteins in tylosin-resistant S. xylosus at the mRNA level. The data displayed that ribosomal protein L23 (rplw), thioredoxin(trxA) and Aldehyde dehydrogenase A(aldA-1) are up-regulated in the tylosin-resistant S. xylosus, compared with the tylosin-sensitive strains. CONCLUSION: Our findings demonstrate the important of stress-response and transcription in the tylosin resistance of S. xylosus and provide an insight into the prevention of this resistance, which would aid in finding new medicines .

Study on microwave assisted extraction of chrysophanol and its intervention in biofilm formation of Streptococcus suis.

A microwave assisted extraction technology was used to extract chrysophanol from rhubarb. The present study will focus on the optimum extraction conditions of chrysophanol and discuss the inhibitory effect of chrysophanol on the biofilm formation of Streptococcus suis (S. suis). A Box-Behnken design based on single-factor experiments was applied to optimize the microwave assisted extraction process and to study the factors' relationships with each other. The results showed that a microwave temperature of 56 °C, ethanol concentration of 70%, microwave power of 540 W and liquid to raw material ratio of 55 mL g-1 were the optimal conditions for the microwave method. The yield of chrysophanol was 2.54 ± 0.07% under the optimal conditions, which was in agreement with the predicted value (2.64%). Then, the chemical structure of the extracted chrysophanol was identified by LC-MS. In addition, in vitro experiments showed that chrysophanol has an inhibitory effect on S. suis (minimum inhibitory concentration was 1.98 µg mL-1) and was shown to significantly inhibit the capability of S. suis to form a biofilm using crystal violet staining. Finally, scanning electron microscopy analysis showed that the three-dimensional structure of the biofilm deposited by the S. suis community was destroyed by chrysophanol.

Process optimization of Syringa oblata Lindl. by response surface methodology and its effect on Staphylococcus xylosus biofilm.

Syringa oblata Lindl. (S. oblata) is a medicinal plant with effective broad-spectrum antibacterial activity, which can also inhibit Streptococcus suis biofilm formation. The processing of herbal medicine can purify medicinal materials, provide acceptable taste, reduce toxicity, enhance efficacy, influence performance and facilitate preparation. Thus, the aim of this study was to enhance the biofilm inhibition activity of S. oblata toward Staphylococcus xylosus (S. xylosus) using the best processing method. The content of rutin and flavonoids and the ability to inhibit the biofilm formation by S. oblata were examined using four processing methods. One of the best methods, the process of stir-frying S. oblata with vinegar, was optimized based on the best rutin content by response surface methodology. The histidine content and hisB gene expression of S. xylosus biofilm in vitro, resulting from stir-frying S. oblata with vinegar, were evaluated and were found to be significantly decreased and down-regulated, respectively. The results show that S. oblata stir-fried with vinegar can be used to effectively treat diseases resulting from S. xylosus infection. This is because it significantly inhibited S. xylosus biofilm formation by interfering with the biosynthesis of histidine; thus, its mechanism of action is decreasing histidine synthesis.

Azithromycin Inhibits Biofilm Formation by Staphylococcus xylosus and Affects Histidine Biosynthesis Pathway.

Staphylococcus xylosus, a coagulase-negative, non-pathogenic bacterium, responsible for opportunistic infections in humans and bovine mastitis, has the ability to form biofilms, which are responsible for persistent infections and antibiotic resistance. In our study, azithromycin significantly inhibited biofilm formation by altering protein expression. Of the 1764 proteins measured by the isobaric Tag for Relative and Absolute Quantification (iTRAQ) technique, only 148 proteins showed significantly different expression between the azithromycin-treated and untreated cells. Most ribosomal proteins were markedly up-regulated, and the expression of the proteins involved in histidine biosynthesis, which, in turn, influence biofilm formation, was down-regulated, particularly imidazole glycerophosphate dehydratase (IGPD). Previously, we had observed that IGPD plays an important role in biofilm formation by S. xylosus. Therefore, hisB expression was studied by real-time PCR, and the interactions between azithromycin and IGPD were predicted by molecular docking analysis. hisB was found to be significantly down-regulated, and six bond interactions were observed between azithromycin and IGPD. Many active atoms of azithromycin did not interact with the biologically active site of IGPD. Surface plasmon resonance analysis used to further study the relationship between IGPD and azithromycin showed minimum interaction between them. Histidine content in the azithromycin-treated and untreated groups was determined. We noted a slight difference, which was not consistent with the expression of the proteins involved in histidine biosynthesis. Therefore, histidine degradation into glutamate was also studied, and we found that all proteins were down-regulated. This could be the reason why histidine content showed little change between the treated and untreated groups. In summary, we found that azithromycin is a potential inhibitor of S. xylosus biofilm formation, and the underlying mechanism was preliminarily elucidated in this study.

Spectrum-Effect Relationships Between the Bioactive Ingredient of Syringa oblata Lindl. Leaves and Its Role in Inhibiting the Biofilm Formation of Streptococcus suis.

Syringa oblata Lindl. (S. oblata) has been used in herbal medicines for treating bacterial diseases. It is also thought to inhibit Streptococcus suis (S. suis) biofilm formation. However, due to the inherent nature of the complexity in its chemical properties, it is difficult to understand the possible bioactive ingredients of S. oblata. The spectrum-effect relationships method was applied to screen the main active ingredients in S. oblata obtained from Heilongjiang Province based on gray relational analysis. The results revealed that Sub-MICs obtained from 10 batches of S. oblata could inhibit biofilm formation by S. suis. Gray relational analysis revealed variations in the contents of 15 main peaks and rutin was discovered to be the main active ingredient. Then, the function of rutin was further verified by inhibiting S. suis biofilm formation using crystal violet staining. Computational studies revealed that rutin may target the chloramphenicol acetyltransferase protein in the biofilm formation of S. suis. In conclusion, this study revealed that the spectrum-effect relationships and computational studies are useful tools to associate the active ingredient with the potential anti-biofilm effects of S. oblata. Here, our findings would provide foundation for the further understanding of the mechanism of S. oblata intervention in biofilm formation.