Antimicrobial and antibiofilm effects of essential fatty acids against clinically isolated vancomycin-resistant Enterococcus faecium.

Introduction: Enterococcus faecium is a leading cause of hospital-acquired infections, which has become a serious public health concern. The increasing incidence of vancomycin-resistant E. faecium (VRE-fm) raises an urgent need to find new antimicrobial agents as a complement to traditional antibiotics. The study aimed to evaluate the antimicrobial and antibiofilm activity of essential fatty acids (EFAs) against VRE-fm, and further explore the molecular mechanism of the antibiofilm activity of EFAs. Method: The microdilution broth method was used for antimicrobial susceptibility testing with traditional antibiotics and EFAs, including α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LOA), γ-linolenic acid (GLA), and arachidonic acid (AA). The effect of EFAs on cell morphology of VRE-fm was investigated by scanning electron microscopy. The crystal violet method was used to evaluate the antibiofilm activities of EFAs against VRE-fm. Furthermore, the expression of biofilm-related genes (acm, atlA, esp, and sagA) of VRE-fm isolates under the action of GLA was analyzed using quantitative reverse transcription PCR (qRT-PCR) assay. Results: VRE-fm isolates were highly resistant to most traditional antibiotics, only highly susceptible to quinupristin-dalfopristin (90.0%), tigecycline (100%), and linezolid (100%). EPA, DHA, and GLA exhibited excellent antimicrobial activity. The MIC50/90 of EPA, DHA, and GLA were 0.5/1, 0.25/0.5, and 0.5/1 mM, respectively. SEM imaging showed that strain V27 adsorbed a large number of DHA molecules. Furthermore, all EFAs exhibited excellent inhibition and eradication activities against VRE-fm biofilms. The biofilm inhibition rates of EFAs ranged from 45.3% to 58.0%, and eradication rates ranged from 54.1% to 63.4%, against 6 VRE-fm isolates with moderate biofilm formation ability. GLA exhibited remarkable antibiofilm activity against VRE-fm isolates. The qRT-PCR analysis showed that GLA could significantly down-regulate the expression of the atlA gene (P < 0.01) of VRE-fm. Conclusion: DHA showed the strongest antibacterial activity, while GLA showed the strongest antibiofilm effect among the EFAs with antibacterial activity. Our novel findings indicate that the antibiofilm activity of GLA may be through down-regulating the atlA gene expression in VRE-fm. Therefore, DHA and GLA had the potential to be developed as therapeutic agents to treat infections related to multiple antimicrobial-resistant E. faecium.

Antibiofilm Activity of Essential Fatty Acids Against Candida albicans from Vulvovaginal Candidiasis and Bloodstream Infections.

Purpose: The biofilm formation of Candida albicans is an important virulence factor as it can increase tolerance to conventional antifungal drugs and the host immune system. The study aimed to assess the effect of essential fatty acids (EFAs) against biofilm formation and mature biofilms of C. albicans strains, which were isolated from vulvovaginal candidiasis and candidemia. Methods: The biofilm formation ability of C. albicans and antifungal activities of fluconazole were determined. Additionally, the effects of six EFAs [α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LOA), γ-linolenic acid (GLA), and arachidonic acid (AA)] against C. albicans under planktonic and biofilm conditions were evaluated. Results: 94.1% of C. albicans exhibited biofilm formation capacity, and 98.5% of C. albicans were susceptible to fluconazole. The biofilms of C. albicans were highly resistant to fluconazole with minimum biofilm eradication concentration values ≥ 64 µg/mL. The EFAs attenuated biofilm formation in a dose-dependent manner, and GLA displayed a remarkable inhibitory activity against biofilm formation of C. albicans. In addition, EPA, DHA, and GLA at 0.1 mM could inhibit the biofilm formation of C. albicans without affecting the planktonic growth rate. Notably, EPA and AA at 1 mM had both inhibitory and eradication activities on C. albicans biofilms. Conclusion: This is the first study to directly compare different EFAs for their capacity to affect C. albicans biofilm formation as well as biofilm eradication. These results suggest EPA and AA could serve as potential new antifungal agents for the treatment of clinical infections caused by C. albicans biofilms.

MADS-Box Transcription Factor MadsA Regulates Dimorphic Transition, Conidiation, and Germination of Talaromyces marneffei.

The opportunistic human pathogen Talaromyces marneffei exhibits a temperature-dependent dimorphic transition, which is closely related with its pathogenicity. This species grows as multinucleate mycelia that produce infectious conidia at 25°C, while undergoes a dimorphic transition to generate uninucleate yeast form cells at 37°C. The mechanisms of phenotype switching are not fully understood. The transcription factor madsA gene is a member of the MADS-box gene family. Previously, it was found that overexpression of madsA gene resulted in mycelial growth instead of yeast form at 37°C. In the current study, the madsA deletion mutant (ΔmadsA) and complemented strain (CMA) were constructed by genetic manipulation. We compared the phenotypes, growth, conidiation, conidial germination and susceptibility to stresses (including osmotic and oxidative) of the ΔmadsA with the wild-type (WT) and CMA strains. The results showed that the ΔmadsA displayed a faster process of the yeast-to-mycelium transition than the WT and CMA. In addition, the deletion of madsA led to a delay in conidia production and conidial germination. The tolerance of ΔmadsA conidia to hydrogen peroxide was better than that of the WT and CMA strains. Then, RNA-seq was performed to identify differences in gene expression between the ΔmadsA mutant and WT strain during the yeast phase, mycelium phase, yeast-to-mycelium transition and mycelium-to-yeast transition, respectively. Gene ontology functional enrichment analyses indicated that some important processes such as transmembrane transport, oxidation-reduction process, protein catabolic process and response to oxidative stress were affected by the madsA deletion. Together, our results suggest that madsA functions as a global regulator involved in the conidiation and germination, especially in the dimorphic transition of T. marneffei. Its roles in the survival, pathogenicity and transmission of T. marneffei require further investigation.

Impacts of HBV rtH55R polymerase substitution on viral replication and rtM204I/V resistance to nucleoside/nucleotide antiviral drugs.

BACKGROUND: High genetic variability at the reverse transcriptase (RT) region of HBV could confer resistance to nucleoside/nucleotide analogues (NUCs). The aim of this study was to identify new RT amino acid (AA) substitutions related to NUC resistance. METHODS: HBV RT sequences of genotype C from 501 chronic hepatitis B (CHB) patients were analysed to identify potential RT substitutions related to NUC resistance. In vitro studies without and with NUCs were performed in a HepG2 cell line transfected by clones with RT harbouring wild-type or substituted AA(s) of interest. RESULTS: Among 261 NUC-treated CHB patients, we found a high detection rate of rtM204I/V substitution (30.7% [80/261]). We identified a new substitution of rtH55R, and its detection rate had a significantly increasing trend from 3.8% (9/240) in the untreated group to 7.2% (13/181) or 33.8% (27/80) in the treated group with rtM204 or with rtM204I/V substitutions (P<0.0001). In vitro studies showed that rtH55R had a similar HBV DNA level compared to wild type. The rtH55R+rtM204I clone had a significantly better replication capacity than the rtM204I clone without NUCs (P<0.05). The replication capacity of the rtM204I clone was found to significantly decrease under lamivudine treatment, but this was not found in the rtH55R+rtM204I clone. CONCLUSIONS: We identified a new HBV RT substitution of rtH55R in genotype-C-infected CHB patients. It is frequently found in combination with rtM204I/V substitution under NUC treatment. In vitro studies suggest that it might play some replication compensatory role in rtM204I mutants under lamivudine treatment.

Antimicrobial Compounds Produced by Vaginal Lactobacillus crispatus Are Able to Strongly Inhibit Candida albicans Growth, Hyphal Formation and Regulate Virulence-related Gene Expressions.

The female vaginal environment contains diverse microorganisms, and their interactions play significant roles in health and disease. Lactobacillus species are the predominant vaginal microorganisms in healthy women and relevant as a barrier to defense against pathogens, including Candida albicans. The yeast-to-hyphae transition is believed to be a determinant of C. albicans pathogenesis. In this study, we investigated the effects of vaginal isolates of L. crispatus (seven strains), L. gasseri (six strains), and L. jensenii (five strains) on growth, hyphal formation and virulence-related genes expression of C. albicans ATCC 10231. We found that the L. crispatus showed the most significant antimicrobial activities in microplate-based liquid medium assay (P < 0.05). All seven cell-free supernatants (CFS) from L. crispatus strains reduced the growth of C. albicans by >60%. The effects might be due to their productions of some secretory antimicrobial compounds in addition to H2O2 and organic acids. Furthermore, each of the CFS of Lactobacillus strains was found to significantly suppress the yeast-to-hyphae transition of C. albicans under hyphae-inducing conditions (RPMI 1640 medium supplemented with 10% fetal bovine serum). The hyphae inhibition rates of C. albicans treated by CFS from L. crispatus, L. gasseri, and L. jensenii were 88.3 ± 3.02%, 84.9 ± 6.0%, and 81.9 ± 6.2%, respectively. Moreover, the expression of hyphae-specific genes (ALS3, HWP1, ECE1, EAP1, and SAP5) and transcriptional regulatory genes (EFG1, TEC1, and NRG1) were analyzed using quantitative real-time PCR. The results demonstrated that L. crispatus CFS significantly down-regulated the expression of hyphae-specific genes ALS3 (0.140-fold)), HWP1 (0.075-fold), and ECE1 (0.045-fold), while up-regulated the expression of the negative transcriptional regulator gene NRG1 with 1.911-fold. The antimicrobial compounds from L. crispatus B145 against Candida growth were heat stable and protease resistance, but those against hyphal formation were partially sensitive to the same treatments. Our novel findings suggest that L. crispatus, a dominant Lactobacillus species associated with a healthy vagina, could strongly inhibit C. albicans growth and hyphal formation. L. crispatus might repress the expression of hyphae-specific genes (ALS3, HWP1, and ECE1) in a NRG1-dependent manner. Besides, L. crispatus B145 is highly worthwhile for probiotic investigation.

[Exposure to 1-bromopropane causes dose-dependent neurological abnormalities in workers].

OBJECTIVE: To explore the dose-effect relationship between 1-bromopropane (1-BP) exposure and health effects in workers. METHODS: Occupational field investigations were conducted in 1-BP factories. Ambient 1-BP concentrations were detected with detection tube, and the 8 h time-weighted average individual exposure levels (TWA-8 h) were measured by passive sampler. Workers underwent questionnaire survey, neurological examination, nerve conduction velocity examination, vibration sensation test. routine blood test as well as blood biochemical test. According to TWA values or TWA x duration values, workers were divided into three dose groups for dose-effect relationship analysis. USEPA BMDS 2.1 software was applied to calculate 1-BP benchmark dose (BMD) and its 95% lower limit (BMDL). RESULTS: The TWA-8h concentrations ranged from 0.35 to 535.19 mg/m3 (geo-mean 14.08 mg/m3). Dose-dependent analysis showed that the motor nerve distal latency (linear regression coefficient was 0.066 6), vibration sensation of toes (linear regression coefficient were 0.157 2 and 0.193 9), creatine kinase (linear regression coefficient was -1.05) and thyroid stimulating hormone levels (linear regression coefficient was 0.1024) of 1-BP exposed workers changed in a dose-dependent manner (P < 0.05). BMD calculation based on DL as 1-BP toxic effect endpoint showed that TWA-8h of the BMD values and BMDL values were 50.55 mg/m3 and 30.78 mg/m3, respectively. CONCLUSION: 1-BP causes dose-dependent changes in tibial nerve DL, vibration sensation, CK and TSH levels.

[Effects of 1-bromopropane on neurological and hematological changes of female exposed workers].

OBJECTIVE: To investigate the health effects of 1-bromopropane (1-BP) on female exposed workers. METHODS: Four 1-BP manufacturing plants were investigated. Workers were interviewed with questionnaire and examined with neurobehavioral core test battery, nerve conduction velocity tests of nervus tibialis and nervus suralis, vibration sensation test, hematological and biochemical tests. Ambient 1-BP concentration was measured with detection tube, and time-weighed average levels of individual workers were estimated with passive samplers. RESULTS: 1-BP concentration in the plants ranged from 0 to 402.40 mg/m3 (Geomean 32.19 mg/m3). Time-weighted average exposure levels (TWA-8 h) ranged from 0.35 to 535.19 mg/m3 (Geomean 14.08 mg/m3). Compared with the control group, 1-BP exposed workers showed reduced motor nerve conduction velocity [(44.8 +/- 8.7) m/s] and sensory nerve conduction velocity [(45.5 +/- 4.9) m/s], prolonged distal latency [(7.5 +/- 2.1) ms], reduced toe vibration perception, and altered neurobehavior parameters(POMS vigor, tension, anxiety, confusion) significantly (P < 0.05). As to hematological and biochemical indicators, the exposed workers showed decreased white blood cell count [(5.6 +/- 2.17) x 10(3)/microl], red blood cell count [(3.9 +/- 0.4) x 10(6)/microl], hemoglobin [(121.1 +/- 14.5) g/L] and creatine kinase [(82.0 +/- 27.5) IU/L] (P < 0.05), and increased serum total protein (8.0 +/- 0.5 g/dl), lactate dehydrogenase [(335.2 +/- 356.6) IU/L], thyroid-stimulating hormone [(3.6 +/- 2.3) microIU/ml] and follicle-stimulating hormone levels (18.7 +/- 24.4 mIU/ml) (P < 0.05). CONCLUSION: 1-BP exposure may affect peripheral nerves and central nervous system, and lead to abnormal hematological and biomedical indicators.

Dose-dependent neurologic abnormalities in workers exposed to 1-bromopropane.

OBJECTIVES: To investigate the health effects of 1-bromopropane (1-BP) and its dose-dependency in 1-BP production factories in China. METHODS: Data of 60 female and 26 male workers in three 1-BP factories and the same number of age-, sex-, and region-matched controls were interviewed and examined. The time-weighed average exposure levels of individual workers were estimated. RESULTS: Regression analysis on exposure level showed dose-dependent increase in the distal latency of tibial nerve, threshold for vibration sense in toes, lactate dehydrogenase, thyroid stimulating hormone, and follicle stimulating hormone in female workers. The analysis also showed dose-dependent decrease in sensory nerve conduction velocity of the sural nerve, red blood cell, and hematocrit in female workers. CONCLUSIONS: The results indicate that exposure to 1-BP induces dose-dependent neurotoxicity in female workers.

Reversibility of the adverse effects of 1-bromopropane exposure in rats.

Previous experiments indicated that 1-bromopropane (1-BP), an alternative to chloroflurocarbons, is neurotoxic and inhibits spermiation in the testis. Here we investigated the reversibility of the toxic effects of 1-BP in rats. Male Wistar rats were divided into three equal groups of 24 each and exposed by inhalation to 0, 400 or 1000 ppm of 1-BP for 6 weeks (8 hrs/day, 7 days/week). Eight rats from each group were sacrificed at the end of 6 weeks exposure, and at 4 and 14 weeks after the end of exposure, to assess the recovery processes. We studied sperm count, motility, morphology and testicular histopathology, as well as blood pressure, skin temperature and hindlimb muscle strength. At the end of 6 weeks of exposure to 1000 ppm (0 week recovery), testicular weight, epididymal weight, sperm count and motility were low, morphologically abnormal sperm were increased and spermatogenic cells showed diffuse degeneration. These changes did not show full recovery at 14 weeks recovery, with the exception of the prostate and seminal vesicular weights, which recovered back to control values. At 400 ppm, increased retained spermatids at 0 week recovery returned to normal at 4 weeks recovery. Exposure to 1000 ppm produced sustained reduction of hindlimb muscle strength at 14 weeks recovery, whereas normalization of the skin temperature and blood pressure was noted after transient changes. Our study showed that the effect of 1-BP on spermatogenesis is dose-dependent; low exposure inhibited spermiation and hormone-dependent organ weight reduction and these changes were transient, while a higher dose of 1000 ppm 1-BP caused persistent depletion of spermatogenic cells.

Urinary 1-hydroxypyrene concentrations in Chinese coke oven workers relative to job category, respirator usage, and cigarette smoking.

BACKGROUND: 1-Hydroxypyrene (1-OHP) is a biomarker of recent exposure to polycyclic aromatic hydrocarbons (PAHs). We investigated whether urinary 1-OHP concentrations in Chinese coke oven workers (COWs) are modulated by job category, respirator usage, and cigarette smoking. METHODS: The present cross-sectional study measured urinary 1-OHP concentrations in 197 COWs from Coking plant I and 250 COWs from Coking plant II, as well as 220 unexposed referents from Control plant I and 56 referents from Control plant II. RESULTS: Urinary 1-OHP concentrations (geometric mean, micromol/mol creatinine) were 5.18 and 4.21 in workers from Coking plants I and II, respectively. The highest 1-OHP levels in urine were found among topside workers including lidmen, tar chasers, and whistlers. Benchmen had higher 1-OHP levels than other workers at the sideoven. Above 75% of the COWs exceeded the recommended occupational exposure limit of 2.3 micromol/mol creatinine. Respirator usage and increased body mass index (BMI) slightly reduced 1-OHP levels in COWs (P < 0.1). Cigarette smoking significantly increased urinary 1-OHP levels in unexposed referents (P < 0.005), but had no effect in COWs (P > 0.1). CONCLUSIONS: Chinese COWs, especially topside workers and benchmen, are exposed to high levels of PAHs. Urinary 1-OHP concentrations appear to be modulated by respirator usage and BMI in COWs, as well as by smoking in unexposed referents.

Higher urinary 1-hydroxypyrene concentration is associated with cooking practice in a Chinese population.

Urinary 1-hydroxypyrene (1-OHP) is a biomarker of polycyclic aromatic hydrocarbons (PAHs) exposure. We examined urinary 1-OHP concentrations in 328 non-occupational exposed health subjects (108 females and 220 males). Each subject was questionnaired for the information of cigarette smoking and the habit of cooking practice including the cooking frequency in the kitchen (times per week), range hood opened for cooking, and fuel used for cooking. Concentrations of urinary 1-OHP (geometric mean, mumol/mol creatinine) were significantly higher in females than in males (0.50 versus 0.34, P<0.001). Cooking frequency in the kitchen had significant influence on the urinary 1-OHP concentrations in females. In females who did cooking practice in the kitchen, the urinary 1-OHP excretions were significantly higher in those who did not install the range hood compared to those who always opened the hood when cooking (0.75 versus 0.42, P=0.020). In males, cooking frequency in the kitchen and cigarette smoking were interacted on urinary 1-OHP levels. Those results imply that it is necessary for females with higher frequency of cooking and males with heavy smoking habits to improve their cooking conditions in order to reduce the risks of getting diseases including cancer.

The influence of metabolic gene polymorphisms on urinary 1-hydroxypyrene concentrations in Chinese coke oven workers.

Urinary 1-hydroxypyrene (1-OHP), a biomarker of polycyclic aromatic hydrocarbons (PAHs) exposure, may be influenced by metabolic gene polymorphisms. Such knowledge could benefit us in understanding the inter-individual difference in the mechanism of PAHs-induced carcinogenesis. We investigated the influence of gene polymorphisms on urinary 1-OHP concentrations in 447 coke oven workers from two coking plants in south China. After adjustment for age, plant, level of occupational exposure, body mass index, level of education, alcohol consumption, cigarette smoking and respirator usage, AhR R554K (rs2066853), UGT1A1 -3263T>G (rs4124874) and GSTP1 I105V (rs1695) were associated with urinary 1-OHP excretion with the p-value of 0.053, 0.006 and 0.021, respectively. The concentrations of urinary 1-OHP (Geometric mean, micromol/mol creatinine) in the homozygous major variant carriers and homozygous minor variant carriers for AhR R554K, UGT1A1 -3263T>G and GSTP1 I105V were listed as follows: 4.20 and 5.12, 5.11 and 3.92, 4.93 and 2.91, respectively. GSTT1 present carriers had a significantly higher urinary 1-OHP level than that in null carriers in the case with AhR R554K GA/AA carriers (5.17 vs. 3.64 micromol/mol creatinine, p=0.038), as well as in the case with UGT1A1 -3263T>G TG/GG carriers (5.67 vs. 3.38 micromol/mol creatinine, p=0.001). These results showed that AhR, UGT1A1, GSTP1 and GSTT1 polymorphisms were associated with urinary 1-OHP concentrations in Chinese coke oven workers. No influence was found in the association between urinary 1-OHP and other genetic polymorphisms such as CYP1A1, CYP1A2, CYP1B1, CYP2E1, EPHX1, EPHX2 in this population.

[Comparison on urinary 1-hydroxypyrene concentrations between coke oven workers and non-occupational exposed individuals].

OBJECTIVE: To compare the urinary 1-hydroxypyrene (1-OHP) concentrations between coke oven workers and non-occupational exposed individuals and to investigate the possible impact factors. METHODS: Spot end-of-shift urine samples were collected in 265 coke oven workers and spot morning urine samples in 226 non-occupational exposed individuals. External exposure levels and possible confounding factors were assessed by environmental polycyclic aromatic hydrocarbons (PAH) monitoring and uniform questionnaire, and the urinary 1-OHP concentrations determined by high performance liquid chromatography (HPLC), were used to compare the different urinary 1-OHP levels between different research populations and to explore the effects of several factors such as external exposure levels, smoking habits, alcohol consumptions, age and body mass index (BMI). RESULTS: The urinary 1-OHP concentrations in coke oven workers were significantly higher than those in non-occupational exposed individuals (13.49, 6.56, 1.38 and 0.35 micromol/mol creatinine for topside workers, side-oven workers, bottom side workers and references, respectively, P < 0.001; the same trends for the percentage of the level over 2.3 micromol/mol creatinine: 94.81%, 84.73%, 35.09% and 0.88%, P < 0.001). The control individuals who smoked over 20 cigarettes per day possessed higher urinary 1-OHP concentrations than the non-smokers (adjusted geometric mean: 0.47 and 0.31 micromol/mol creatinine, respectively, P < 0.05). Alcohol drinking references possessed lower urinary 1-OHP levels than the non-drinkers (adjusted geometric mean: 0.33 and 0.47 micromol/mol creatinine, respectively, P < 0.05). CONCLUSION: Coke oven workers should be exposed to a high level of urinary 1-OHP, especially for topside workers and side-oven workers. Background urinary 1-OHP levels in non-occupational exposed individuals should be related to smoking habits and alcohol consumptions.