The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis

Macroautophagy/autophagy is a central mechanism by which cells maintain integrity and homeostasis, and endotoxin-induced autophagy plays important roles in innate immunity. Although TLR4 stimulation mediated by lipopolysaccharide (LPS) also upregulates autophagy in hepatocytes and liver, its physiological role remains elusive. The objective of this study was to determine the role of LPS-induced autophagy in the regulation of liver lipid metabolism. LPS treatment (5 mg/kg) increased autophagy, as detected by LC3 conversion and transmission electron microscopy (TEM) analysis in C57BL6 mouse livers. AC2F hepatocytes also showed increased autophagic flux after LPS treatment (1 μg/ml). To investigate the role of LPS-induced autophagy further, liver lipid metabolism changes in LPS-treated mice and fasted controls were compared. Interestingly, LPS-treated mice showed less lipid accumulation in liver than fasted mice despite increased fatty acid uptake and lipid synthesis-associated genes. In vitro analysis using AC2F hepatocytes demonstrated LPS-induced autophagy influenced the degradation of lipid droplets. Inhibition of LPS-induced autophagy using bafilomycin A₁ or Atg7 knockdown significantly increased lipid accumulation in AC2F hepatocytes. In addition, pretreatment with chloroquine aggravated LPS-induced lipid accumulation and inflammation in C57BL6 mouse livers. The physiological importance of autophagy was verified in LPS-treated young and aged rats. Autophagic response was diminished in LPS-treated aged rats and lipid metabolism was impaired during sepsis, indicating autophagy response is important for regulating lipid metabolism after endotoxin challenge. Our findings demonstrate endotoxin-induced autophagy is important for the regulation of lipid metabolism, and suggest that autophagy helps maintain lipid metabolism homeostasis during sepsis.     

Distribution of Pathogenic Genes aatA, aap, aggR, among Uropathogenic Escherichia coli (UPEC) and Their Linkage with StbA Gene

Urinary tract infection (UTI) with E. coli (UPEC) is one of the most common bacterial infections among human beings. In addition to the host predisposing factors, genes are also proposed to have an important role in the occurrence of UTIs. This study investigated the distribution of three pathogenic genes including aggR, aap and aatA among UPEC infected samples and their linkage with stbA, the essential gene for maintaining of pAA plasmid. A total of 244 samples were collected from patients with UTIs through clinical laboratories located in western side of Tehran (Iran) during years 2008–2009. E. coli isolation was performed according to standard laboratory methods. DNAs were extracted from samples using Boiling method, and the presence of aap, aggR, aatA and stbA genes were investigated by PCR. No pathogenic genes (aap, aggR, aatA) were found in 104 out of 244 UPEC samples, while 14 of them were carrying stbA gene. Out of 140 UPEC samples with pathogenic genes, 94 (46.6%) were carrying aap gene, 52 (23%) aggR gene, and 80 (35.4%) aatA gene. A total of 18 samples were also carrying all pathogenic genes together. Moreover, 44 out of 144 samples were carrying stbA gene. The results obtained by this study showed that the aggR, aap and aatA pathogenic genes have different existence patterns in different E. coli strains that infect different organs. Our study also showed that these three plasmid genes in EAEC strains are able to transpose in the genome and change their level of linkage with pAA plasmid essential gene stbA. Meanwhile, this study confirmed that aggR, aap and aatA genes are not specific to only EAEC strains.     

Use of Pyromark Q96 ID pyrosequencing system in identifying bacterial pathogen directly with urine specimens for diagnosis of urinary tract infections

Detection and identification of bacterial etiology in urine is critical for accurate diagnosis and subsequent rational treatment of urinary tract infections (UTIs). Urine culture followed by a series of biochemical reactions is currently the standard method for detecting and distinguishing microorganisms associated with UTIs. The whole procedure commonly takes more than 24 h. Here we developed a new system combining 16S rRNA gene broad-range PCR with pyrosequencing technology that allows for bacteria detection and identification in urine in 5 h. To evaluate this system for rapid diagnosis of bacteriuria, 768 urine specimens were collected from patients with suspected UTIs and were tested side-by-side using standard urine culture-based identification method and the pyrosequencing method. The results from pyrosequencing correlated well with those from traditional culture-based identification method. The overall agreement between these two methods reached 98.0% (753/768). In addition, we tested the sensitivity of pyrosequencing method and determined that urine bacterial numbers as low as 10⁴ cfu/ml could be accurately detected and identified. In conclusion, compared with traditional biochemical method, the PCR-pyrosequencing system significantly improved the detection and identification of bacteriuria with shorter time, higher accuracy, and higher throughput, thus allowing earlier pathogen-adapted antibiotic therapy for patients.     

The molecular dissection of the chaperone–usher pathway

Secretion systems are specialized in transport of proteins, DNA or nutrients across the cell envelope of bacteria and enable them to communicate with their environment. The chaperone–usher (CU) pathway is used for assembly and secretion of a large family of long adhesive protein polymers, termed pili, and is widespread among Gram-negative pathogens [1]. Moreover, recent evidence has indicated that CU secretion systems are also involved in sporulation  and . In this review we focus on the structural biology of the paradigmatic type 1 and P pili CU systems encoded by uropathogenic Escherichia coli (UPEC), where recent progress has provided unprecedented insights into pilus assembly and secretion mechanism. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

CysC,mALB和尿BGM联合检测在2型糖尿病早期肾损中的临床价值

目的:探讨CysC、mALB和尿BGM联合检测在2型糖尿病早期肾损中的临床价值。方法:选取2010年11月至2013年6月我院内分泌科收治的130名2型糠尿病患者,根据空腹血糖值的不同程度将其分为A、B两组,A组72例,B组58例,另选取同期在我院体检的年龄、性别等资料与之匹配的64名健康者设为C组。分别对其CysC、mALB和尿BGM进行测定,并进行对比分析。结果:通过分析可知,A组和B组的Cys C分别为(3.29±1.09)mg/L、(2.86±1.05)mg/L,相对于C组的(0.87±0.99)mg/L,均明显升高,但A组升高更加明显;A组、B组、C组的mALB分别为(37.36±4.82)mg/L、(36.55±4.31)mg/L、(6.61±3.84)mg/L,B组高于A组,C组高于A组和B组;三组的BGM中,以A组最高,为(634.15±55.24)μg/L,B组其次,为(626.92±48.18)μg/L,均高于C组的(57.12±11.75)μg/L;Cys C、mALB、BGM等阳性指标的检出率分别为76.15%、77.69%、72.31%,而Cys C、mALB、BGM联合检测的检出率为93.85%,明显高于其他单指标检测。且P0.05,差异具有统计学意义。结论:对CysC、mALB和尿BGM进行联合检测对2型糖尿病早期肾损患者具有较高的检出率,相对于单指标检测,敏感性更高。     

内毒素和降钙素原在妇科术后尿路感染中的鉴别诊断价值

目的评价中段尿内毒素和血清降钙素原在妇科术后不同种类细菌尿路感染中的鉴别诊断价值。方法收集临床1205例妇科术后患者中段尿进行细菌培养及内毒素检测,同时对患者进行血清降钙素原检测,比较结果对尿路感染的鉴别诊断价值。结果1205份标本中尿培养出阳性350例,感染率为29．04％,其中298例为均存在留置导尿管,而在剩余400例尿培养阴性的患者中仅仅120例留置导尿管。两组之间差异有统计学意义（χ2=26．78,P〈0．05）。其中革兰阴性杆菌189例（54％）,革兰阳性菌112例（32％）,真菌49例（14％）。在三组患者中,中段尿内毒素在革兰阴性菌引起的术后尿路感染较革兰阳性菌和真菌的患者中明显升高,差异均有统计学意义（P〈0．05）。而对于血清降钙素原在革兰阴性菌和革兰阳性菌感染的患者明显高于真菌尿路感染的患者,差异均有统计学意义（P〈0．05）。而在革兰阴性菌和革兰阳性菌感染的患者中差异无统计学意义（P〉0．05）。结论妇科术后尿路感染与留置导尿管密切相关,革兰阴性菌是引起妇科术后尿路感染的主要致病菌,中段尿内毒素有助于鉴别诊断出革兰阴性菌引起尿路感染,而血清PCT升高时则有助于排除真菌尿路感染。     

Circulating soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as diagnostic and prognostic marker in neonatal sepsis

ObjectiveTriggering receptor expressed on myeloid cells-1 (TREM-1) is an important receptor involved in the innate inflammatory response and sepsis. We assessed soluble TREM-1 (sTREM-1) in 112 septic neonates (63 culture-positive and 49 culture-negative) and 40 healthy controls as a potential early diagnostic and prognostic marker for neonatal sepsis (NS).MethodsStudied neonates were evaluated for early- or late-onset sepsis using clinical and laboratory indicators upon admission. sTREM-1 was measured on initial sepsis evaluation and at 48 h after antibiotic therapy. For ethical reasons, cord blood samples were collected from control neonates and only samples from neonates that proved to be healthy by clinical examination and laboratory analysis were further analyzed for sTREM-1.ResultsBaseline sTREM-1 levels were significantly elevated in culture-proven (1461.1 ± 523 pg/mL) and culture-negative sepsis (1194 ± 485 pg/mL) compared to controls (162.2 ± 61 pg/mL) with no significant difference between both septic groups. Culture-positive or negative septic preterm neonates had significantly higher sTREM-1 compared to full term neonates. sTREM-1 was significantly higher in neonates with early sepsis than late sepsis and was associated with high mortality. sTREM-1 was significantly decreased 48 h after antibiotic therapy compared to baseline or levels in neonates with persistently positive cultures. sTREM-1 was positively correlated to white blood cells (WBCs), absolute neutrophil count, immature/total neutrophil (I/T) ratio, C-reactive protein (hs-CRP) and sepsis score while negatively correlated to gestational age and weight. hs-CRP and sepsis score were independently related to sTREM-1 in multiregression analysis. sTREM-1 cutoff value of 310 pg/mL could be diagnostic for NS with 100% sensitivity and specificity (AUC, 1.0 and 95% confidence interval [CI], 0.696–1.015) while the cutoff value 1100 pg/mL was predictive of survival with 100% sensitivity and 97% specificity (AUC, 0.978 and 95% CI, 0.853–1.13). However, hs-CRP cutoff 13.5 mg/L could be diagnostic for NS with a sensitivity of 76% and specificity of 72% (AUC, 0.762 and 95% CI, 0.612–0.925) and levels were not related to survival as no significant difference was found between dead and alive septic neonates.ConclusionsElevated sTREM-1 could be considered an early marker for NS that reflects sepsis severity and poor prognosis.     

Yeasts isolated from nosocomial urinary infections: Antifungal susceptibility and biofilm production

BackgroundUrinary Candida infections in the hospital environment are frequent and need to be better understood.AimsTo compare the results of antifungal susceptibility profiles of yeasts isolated from patients with urinary infections obtained by broth microdilution method (BM) and by disk diffusion (DD), and also evaluate the capacity of these yeasts to form biofilms.MethodsOnly yeasts obtained from pure urine cultures with counts higher than 10⁵ colony-forming units per milliliter, without bacteria development, of symptomatic patients were included. The isolates were identified by classical methods and the antifungal susceptibility tests were performed with the following drugs: amphotericin B, ketoconazole, fluconazole, itraconazole, voriconazole and caspofungin. The biofilm studies were carried out in polystyrene microtitration plates.ResultsNinety-five yeasts isolates were analyzed, including 40 Candida albicans, 31 Candida glabrata, 24 Candida tropicalis. In general, the majority of the isolates were susceptible to the tested drugs but some resistance was observed, especially against fluconazole. Great variability in the antifungal susceptibility results was observed with the different tested drugs and a few discrepancies were observed between both methods. We suggest that in case of DD resistance this result should be confirmed by BM, the standard method. C. tropicalis isolates showed high biofilm production (91.7%) compared to C. albicans (82.5%) and C. glabrata (61.3%), with statistical significance (p = 0.0129).ConclusionsCandiduria in critical patients requires major attention and a better control. The different susceptibility results obtained in this study showed the need to identify yeasts up to the species level, especially in patients with urinary tract infection. The development of techniques of antifungal susceptibility tests can help the clinicians in the empiric treatment of candiduria.     

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.