Interaction of biofilm and efflux pump in clinical isolates of carbapenem resistant P. aeruginosa.

OBJECTIVE: Carbapenem-resistant P. aeruginosa (CRPA) is particularly worrisome because of its resistance against multiple antimicrobial agents which reduces treatment options. The efflux pump decreases antibiotic abundance, and biofilm impairs the penetration of antibiotics. The aim of the present study was to evaluate the role and relationship of efflux pump and biofilm formation in CRPA isolates obtained from different clinical samples. PATIENTS AND METHODS: A total of 110 different clinical samples were collected from three tertiary medical hospitals. The samples were subjected to isolation and identification by standard operating procedures. Species level were identified using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system. Antibiotic susceptibility testing was done by broth microdilution method. Crystal violet (CV) staining for observing the biofilm forming ability and amplification of efflux pump mexA gene were also performed on clinical CRPA isolates. Three efflux pump MexAB-OprM regulatory genes were analyzed using sequencing methods. The expression of mexA gene both in biofilm and planktonic bacteria was observed by Quantitative real-time PCR (qRT-PCR). RESULTS: The results showed that 110 samples were CRPA and among them 83 (75.5%) were MDR isolates. The CV staining showed 105 (95.5%) isolates as biofilm producers while 78 (74.3%) MDR isolates showed biofilm formation. mexA hyperexpression was detected in 27 (24.5%) CRPA isolates while 26 (96.3%) in biofilm forming isolates and 96.3% (26/27) in MDR P. aeruginosa. Multiple mutations in nalC, nalD, and mexR genes were detected. The distinct difference confirmed that the expression of mexA gene in P. aeruginosa biofilm producer was significantly higher than that of planktonic bacteria in vitro, and the efflux pump inhibitor PAßN significantly inhibited biofilms in CRPA isolated from clinical samples. CONCLUSIONS: The biofilm and efflux pumps might be two intertwined processes involved in CRPA isolates. Their synergistic effect magnified the drug resistance characteristics of P. aeruginosa.

Two polyurethanases PueA and PueB are major extracellular lipases partly secreted by the mediation of their cognate ABC exporter AprDEF in Pseudomonas protegens Pf-5.

Two polyurethanases PueA and PueB from Pseudomonas protegens Pf-5 have been reported to have hydrolytic activity against synthetic p-nitrophenyl palmitate of lipase substrate, and PueA may play a more effective role in this activity. However, it is still unknown whether PueA and PueB play similar parts in the lipase activity against natural acylglycerols and achieve the extracellular secretion via their cognate ABC exporter AprDEF. In this study, we investigated these questions through the construction of four markerless deletion mutants in Pf5139 (Δupp derivative of Pf-5), two heterologous co-expression strains and their three control strains in lipase-free Escherichia coli BL21(DE3), and detected their lipase activities by the tributyrin plate assay and the liquid culture assay. The results showed that PueA and PueB, classified as subfamily I.3 lipases, are major extracellular lipases involved in the uptake of oil in Pf-5, and PueA plays a leading role in extracellular lipase activity. In addition, the extracellular secretion of PueA and PueB can be partly mediated via AprDEF in Pf-5 and BL21(DE3). Finally, PueA and PueB are also able to achieve the extracellular secretion without the assistance of AprDEF in Pf-5 and BL21(DE3).

Evaluation of the overall impact of antibiotics growth promoters on broiler health and productivity during the medication and withdrawal period.

The effectiveness of some common combination of antibiotic growth promoters (AGP) on growth performance, gut health, and meat quality was evaluated during the medication and withdrawal period in broilers. A total of 540 male Arbor Acre broilers at 0 D of age were randomly assigned to 5 treatments, with 6 replicates of 18 chicks. Broilers received diets during the medication period (0 to 42 D) as follows: NC (control diet without AGP), EN (NC + enduracidin 8 ppm + colistin sulfate 8 ppm), BZ (NC + bacitracin zinc 40 ppm + colistin sulfate 8 ppm), CT (NC + chlortetracycline 50 ppm + colistin sulfate 8 ppm), and VG (NC + virginiamycin 20 ppm + colistin sulfate 8 ppm). Broilers were switched to the same finisher diet without AGP during the withdrawal period (43 to 49 D). The feed:gain ratio in EN, BZ, CT, and VG groups were significantly decreased by 0.07, 0.10, 0.06, and 0.05 during 0 to 42 D (P < 0.05), but increased by 0.19 (P > 0.05), 0.33 (P > 0.05), 0.49 (P < 0.05), and 0.69 (P < 0.05) during the withdrawal period, respectively. The jejunum villus height (VH) increased in EN group (P < 0.05) and crypt depth (CD) reduced in BZ, CT, and VG groups (P < 0.05) at 42 D, while jejunum VH increased in EN and BZ groups (P < 0.05) at 49 D compared to NC group (P < 0.05). Meat quality detection at 49 D found all AGP groups with the higher cook loss of the breast muscle, while CT group with the higher cook loss of thigh muscle. Consequently, the overall effects of 4 AGP combinations in the whole period were not significant on growth performance. Their poor growth performance during the withdrawal period should be partly attributed to the falling off a cliff of most digestive enzyme activities from 42 to 49 D. Attention should be paid to the adverse effects of AGP supplementation on meat quality, especially cook loss.

Influence of acidified drinking water on growth performance and gastrointestinal function of broilers.

The ban on the use of antibiotic feed additives as growth promoters compelled the researchers for exploring the future utility of other alternatives. This experiment was designed to evaluate the effect of acidified drinking water on growth performance, gastrointestinal pH, digestive enzymes, intestinal histomorphology, and cecum microbial counting of the broiler chicken. A total of 540 one-day-old male broilers (Arbor Acre) were randomly assigned to 5 treatments, with 6 replicates of 18 chicks per replicate. Broilers received diets and water as follows: NC (negative control, basal diet, normal water), PC (positive control, basal diet + 8 ppm colistin sulfate + 8 ppm enduracidin, normal water), A1 (basal diet, continuous supply of acidified water during whole experiment period), A2 (basal diet, intermittent acidification of water during 0 to 14 d, 22 to 28 d, and 36 to 42 d), and A3 [basal diet, intermittent acidification of water (24 h/d from 0 to 14 d and from 10:00 am to 4:00 pm on d 15 to 42)]. During the entire period, the acidified groups (A1, A2, and A3) and PC group showed improve on weight gain, average daily gain and feed conversion ratio compared to NC group (P < 0.05). The pH in crop, proventriculus and ileum at 43 d declined by 0.04, 1.03, 1.23; 0.55, 0.69, 0.70; and 0.63, 0.74, 1.21 in A1, A2, and A3 group, respectively. There was a significant decline of lipase activity in the PC and acidified groups compared to NC group. The A2 group had higher villus height in jejunum than NC group. The PC and acidified groups reduced (P < 0.05) the total aerobic bacteria count of cecum when contrasted to NC group. Therefore, we conclude that acidified drinking water can improve growth performance, compensate for gastric acidity, and control pathogenic bacteria in broilers and may be considered as a potential alternative to improve production parameters. Discontinuous supply of acidified water had the same or even better influence on broilers compared to continuous supply.

Population pharmacokinetics and Bayesian estimation of tacrolimus exposure in Chinese liver transplant patients.

WHAT IS KNOWN AND OBJECTIVES: Tacrolimus (TAC) is widely used as part of immunosuppressive regimens. There is great interindividual variation on the disposition of TAC. The aim of this study was to develop a population pharmacokinetic (PPK) model for Chinese liver transplant patients and evaluate genetic polymorphism and other possible factors on the PK parameters. The exposure of TAC is to be estimated through Bayesian modelling. METHODS: A total of 47 sets of rich-time PK and 1234 conventional therapeutic drug monitoring (TDM) data were collected from 125 Chinese liver transplant patients. The pathophysiological data of these patients were recorded. CYP3A5*3 and ABCB1 genotypes were determined for each patient. The PPK model for TAC was established by nonlinear mixed-effects modelling (nonmem). The impact of pathophysiology and genotype on PPK parameters was evaluated. Bayesian estimators for the area under concentration-time curve (AUC) of TAC were validated. RESULTS: A two-compartment model with lag time was found to be the most suitable model for the pooled full PK and TDM data for Chinese liver transplant patients. The CL/F, V2 /F, Q/F, V3 /F, Ka and lag time were 17.4±0.81 L/h, 165±44.1 L, 54.9±25.8L/h, 594±87.5 L, 0.51±0.095 L/h and 1.57±0.34 h. Post-operative day (POD), creatinine clearance (CLcr) and ABCB1 C3435T genotypes were found to have significant influences on CL/F (P<.01). ABCB1 C3435T genotypes showed a significant correlation with V2 /F (P<.01). C0 -C2 and C0 -C2 -C4 were shown to be suitable for the estimation of AUC in Chinese liver transplant patients. WHAT IS NEW AND CONCLUSION: A PPK model for TAC was established successfully in Chinese liver transplant patients. POD, CLcr and ABCB1 C3435T genotypes were shown to have significant effects on CL/F. The AUC of TAC in Chinese liver transplant patients could be estimated through Bayesian modelling, based on which individualized immunosuppressive regimens can be designed.

Identification of 19 loci for reproductive traits in a local Chinese chicken by genome-wide study.

Reproductive traits have long been studied and have an important influence on chicken breeding. To identify quantitative trait loci affecting reproductive traits, a genome-wide analysis of a Chinese chicken breed was performed to analyze age at first egg body weight at first egg, first egg weight, egg weight at the age of 300 days, egg weight at the age of 462 days, egg number at the age of 300 days, egg number between the ages of 300 and 462 days and egg number at the age of 462 days. Nineteen SNPs related to reproductive traits were presented (P < 1.80E-6). Nine of the 19 SNPs had a significant effect on BWF, six SNPs were significantly associated with egg weight, and four SNPs were significantly associated with egg number. These SNPs were located near to or in 17 genes including FAM184B, HTT, KCNH7, CDC42BPA, KCNIP4, GJA5, CBFB, and GPC6. The present results may be beneficial for reproductive research and may be used in marker-assisted selection in future studies. These results could potentially benefit further breeding programs, especially in Jinghai Yellow Chicken.

Genome-wide association study on reproductive traits in Jinghai Yellow Chicken.

To identify molecular markers and candidate genes associated with reproductive traits, a genome-wide analysis was performed in Jinghai Yellow Chickens to analyze body weight at first oviposition (BWF), age at first oviposition (AFE), weight of the egg at first oviposition (FEW), egg weight at the age of 300 days (EW300), number of eggs produced by 300 days of age (EN300), egg hatchability (HA) and multiple selection index for egg production (MSI). The results showed that seven single nucleotide polymorphisms (SNPs) were associated with reproductive traits (P<1.80E-6, Bonferroni correction). The P-values of the seven SNPs were 5.62E-10, 3.45E-08, 9.76E-07, 8.90E-07, 1.12E-06, 1.42E-07 and 1.48E-07, respectively. These SNPs were located in close proximity to or within the sequence of the five candidate genes, including FAM184B, TTL, RGS1, FBLN5 and PCNX. An additional 46 SNPs that could be associated with reproductive traits were identified (P<3.59E-5, Bonferroni correction). Identification of the candidate genes as well as genome-wide SNPs that may be associated with reproductive traits will greatly advance the understanding of the genetic basis and molecular mechanisms underlying reproductive traits and may have practical significance in breeding programs for the improvements of reproductive traits in the Jinghai Yellow Chicken.

miR-499A>G rs3746444 and miR-146aG>C expression and hepatocellular carcinoma risk in the Chinese population.

We conducted a case-control study of a possible association of miR-499A>G rs3746444 and miR-146aG>C rs2910164 with risk of hepatocellular carcinoma. Samples from 172 hepatocellular carcinoma patients and 185 cancer-free controls were collected from October 2008 to December 2011. PCR-RFLP analysis was performed to determine the polymorphisms in each individual. The MAFs of miR-146aG>C and miR-499A>G in controls were similar to that known from the SNP database, and frequencies of genotypes in controls were in line with Hardy-Weinberg equilibrium. We found that miR-499 AG was significantly associated with decreased risk for hepatocellular carcinoma when compared with miR-499 AA genotype (adjusted odds ration = 0.74, 95% confidence interval = 0.24-0.96). However, subjects carrying miR-146a GG had a non-significant 0.62-fold decreased risk of hepatocellular carcinoma. We did not find a significant association of miR-146aG>C rs2910164 and miR-499A>G rs3746444 polymorphisms with hepatocellular carcinoma risk in the Chinese population. Further investigations are warranted to clarify the relationship between miRNA polymorphisms and susceptibility to hepatocellular carcinoma risk in various ethnic populations.

Single P and As dopants in the Si(001) surface.

Using first-principles density functional theory, we discuss doping of the Si(001) surface by a single substitutional phosphorus or arsenic atom. We show that there are two competing atomic structures for isolated Si-P and Si-As heterodimers, and that the donor electron is delocalized over the surface. We also show that the Si atom dangling bond of one of these heterodimer structures can be progressively charged by additional electrons. It is predicted that surface charge accumulation as a result of tip-induced band bending leads to structural and electronic changes of the Si-P and Si-As heterodimers which could be observed experimentally. Scanning tunneling microscopy (STM) measurements of the Si-P heterodimer on a n-type Si(001) surface reveal structural characteristics and a bias-voltage dependent appearance, consistent with these predictions. STM measurements for the As:Si(001) system are predicted to exhibit similar behavior to P:Si(001).

Animal model of coronary artery spasm-coronary thrombosis-acute myocardial infarction. A study on hemodynamics, EKG, coronary angiography, biochemistry and pathology.

An animal model of coronary artery spasm-coronary thrombosis-acute myocardial infarction (CAS-CATH-AMI) was obtained by injecting ergonovine(0.22 mg/kg) directly into the left coronary artery (LCA) of 17 dogs under general anesthesia. Various parameters of the experimental group were compared with those of the control group consisting of 5 dogs. The following changes were observed: increased average arterial blood pressure (MAP), pulmonary capillary wedge pressure (PCWP), stroke volume (SV, P less than 0.05), PAP (P less than 0.01) transient decreased cardiac output (CO); elevated ST-T in EKG; ventricular arrhythmia in 60% of animals; transient spasm of 50%-75% of LCA in the LCA angiographs; enhancement of platelet aggregation and TXB2 (P less than 0.01) and decline of 6 Keto-PGF1 alpha, SAO2 (P less than 0.05), indicating acute hypoxia and high coagulating pathophysiological changes after CAS. Pathological examination one hour after CAS induction revealed CATH(53%) in addition to CAS induced morphological changes of the CA and myocardium, as well as necrosis of the corresponding sites in its early stage. This study provides a reproducible animal model of CAS-CATH-AMI for research of coronary heart disease and pathomorphological criteria for the diagnosis of CAS. It also shows that CAS may lead to CATH-AMI, therefore, prevention of CATH and AMI is possible.

Protein extraction with reverse micelles.

Liquid-liquid protein extraction with reverse micelles is a new method for protein separation. We studied the effects of pH, ionic concentration, cation type, and protein molecular mass on performance of an AOT/isooctane reverse micelle system in extraction of lysozyme, trypsin, and pepsin. Extraction was found to be very efficient for low-molecular-mass proteins.

[Glomus tumor of the stomach--a case report on a light and electron microscopic study].

This paper reports a case of glomus tumor of stomach from a male patient, 58 years old, with the chief complaint of nausea and vomiting after meal. This tumor was at the anterior wall of the antrum near the lesser curvature. By light microscopy, it was composed of glomus tumor cells and abundant blood vessels. Ultrastructurally, there were numerous filaments, dense bodies, adhesive bodies and pinocytotic vesicles in the cytoplasm. This kind of tumor should be differentiated from the leiomyoblastoma and hemangiopericytoma.

[Pulmonary oncocytic carcinoid--a case report].

A pulmonary oncocytic carcinoid arising from the medial basal segment of the right lung is presented. The abundant oncocytic granules in the cytoplasm were observed by light-microscope. The ultrastructure by electronmicroscope showed that these granules originated from the hyperplasia of mitochondria, and there were membrane-bordered electric dense core granules (neuro-secretive granule) in the cytoplasm of this tumor. According to the morphological features observed by light and electronmicroscope, this tumor is considered as a subtype of bronchial carcinoid and belongs to the APUD system which should be differentiated from the metastatic tumor, the granular cell myoblastoma and the chemodectoma of lung.