Membrane vesicles (MVs) are non-replicating spherical nanoparticles produced by bacteria. The MVs actively released from Gram-negative bacteria are termed outer membrane vesicles (OMVs). OMVs carry various biomolecules, such as lipopolysaccharides, peptidoglycans, proteins, and nucleic acids for material exchange between cells and perform component-dependent physiological functions. In recent years, OMVs have been developed into various biological products, such as vaccines, adjuvants, drug delivery carriers, and cancer immunotherapy agents because of their unique structures and functions. This review describes the biogenesis, composition, and physiological functions of OMVs of Gram-negative bacteria, summarizes the recent research progress of OMVs in product development and cell modifications or engineering, highlights new methods for OMV preparation and characterization, and provides an outlook on the future research directions, with the aim to provide a good reference for study and development of the application of OMVs in the biomedical field.
Gram-Negative Bacteria/metabolism* ; Bacterial Outer Membrane/metabolism* ; Bacterial Outer Membrane Proteins/metabolism* ; Lipopolysaccharides/metabolism*

Extracellular vesicles (EVs), also known as membrane vesicles, are vesicular bodies secreted by eukaryotic cells and bacteria. EVs can carry proteins, DNA, RNA, and various metabolites for the exchange and transmission of substances between cells. They play contents-dependent physiological functions, such as delivering nutrients, participating in immune response, and treating cancers. Currently, most studies focus on the exploration of vesicles secreted by eukaryotic cells and gram-negative bacteria, while few studies focus on gram-positive bacteria. This review summarized the production, content composition, physiological function, and engineering of EVs secreted by gram-positive bacteria, and prospected future perspectives in this area.
Bacteria/metabolism* ; Extracellular Vesicles/metabolism* ; Gram-Negative Bacteria ; Gram-Positive Bacteria/metabolism* ; Proteins/metabolism*

BACKGROUND: Ciprofloxacin is usually used in the treatment of lower respiratory tract infections (LRTIs). Recent studies abroad have shown ciprofloxacin is inadequately dosed and might lead to worse outcomes. The aim of this study was to perform pharmacokinetic and pharmacodynamic analyses of ciprofloxacin in elderly Chinese patients with severe LRTIs caused by Gram-negative bacteria. METHODS: From September 2012 to June 2014, as many as 33 patients were empirically administered beta-lactam and ciprofloxacin combination therapy. Patients were infused with 200 or 400 mg of ciprofloxacin every 12 h, which was determined empirically by the attending physician based on the severity of the LRTI and the patient's renal condition. Ciprofloxacin serum concentrations were determined by high-performance liquid chromatography. Bacterial culture was performed from sputum samples and/or endotracheal aspirates, and the minimum inhibitory concentrations (MICs) of ciprofloxacin were determined. The ratios of the area under the serum concentration-time curve to the MIC (AUC/MIC) and of the maximum serum concentration of the drug to the MIC (Cmax/MIC) were calculated. The baseline data and pharmacokinetic parameters were compared between clinical success group and clinical failure group, bacteriologic success group and bacteriologic failure group. RESULTS: Among the 33 patients enrolled in the study, 17 were infected with Pseudomonas aeruginosa, 14 were infected with Acinetobacter baumannii, and two were infected with Klebsiella pneumoniae. The mean age of the patients was 76.9 ± 6.7 years. Thirty-one patients (93.4%) did not reach the target AUC/MIC value of >125, and 29 patients (87.9%) did not reach the target Cmax/MIC value of >8. The AUC/MIC and Cmax/MIC ratios in the clinical success group were significantly higher than those in the clinical failure group (61.1 [31.7-214.9] vs. 10.4 [3.8-66.1], Z = -4.157; 9.6 [4.2-17.8] vs. 1.3 [0.4-4.7], Z = -4.018; both P < 0.001). The AUC/MIC and Cmax/MIC ratios in the patients for whom the pathogens were eradicated were significantly higher than those in the patients without the pathogens eradicated (75.3 [31.7-214.9] vs. 10.5 [3.8-66.1], Z = -3.938; 11.4 [4.2-17.8] vs. 1.4 [0.4-5.4], Z = -3.793; P < 0.001 for both). Receiver operating characteristic curve analysis showed that the AUC/MIC and Cmax/MIC values were closely associated with clinical and bacteriologic efficacies (P < 0.001 in both). CONCLUSIONS Ciprofloxacin is inadequately dosed against Gram-negative bacteria, especially for those with relatively high MIC values. Consequently, the target values, AUC/MIC > 125 and Cmax/MIC > 8, cannot be reached.
Acinetobacter baumannii ; drug effects ; pathogenicity ; Aged ; Aged, 80 and over ; Chromatography, High Pressure Liquid ; Ciprofloxacin ; pharmacokinetics ; pharmacology ; Female ; Gram-Negative Bacteria ; drug effects ; pathogenicity ; Humans ; Male ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa ; drug effects ; pathogenicity ; Respiratory Tract Infections ; drug therapy ; metabolism ; microbiology

Outer membrane proteins (OMPs) of Gram-negative bacteria constitute the first line of defense protecting cells against environmental stresses including chemical, biophysical, and biological attacks. Although the 43-kDa OMP (OMP43) is major porin protein among Bartonella henselae-derived OMPs, its function remains unreported. In this study, OMP43-deficient mutant B. henselae (Δomp43) was generated to investigate OMP43 function. Interestingly, Δomp43 exhibited weaker proliferative ability than that of wild-type (WT) B. henselae. To study the differences in proteomic expression between WT and Δomp43, two-dimensional gel electrophoresis-based proteomic analysis was performed. Based on Clusters of Orthologus Groups functional assignments, 12 proteins were associated with metabolism, 7 proteins associated with information storage and processing, and 3 proteins associated with cellular processing and signaling. By semi-quantitative reverse transcriptase polymerase chain reaction, increases in tldD, efp, ntrX, pdhA, purB, and ATPA mRNA expression and decreases in Rho and yfeA mRNA expression were confirmed in Δomp43. In conclusion, this is the first report showing that a loss of OMP43 expression in B. henselae leads to retarded proliferation. Furthermore, our proteomic data provide useful information for the further investigation of mechanisms related to the growth of B. henselae.
Bartonella henselae ; Bartonella ; Gram-Negative Bacteria ; Information Storage and Retrieval ; Membrane Proteins ; Membranes ; Metabolism ; Proteomics ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger

No abstract available.
Anti-Bacterial Agents/*pharmacology ; Azabicyclo Compounds/*pharmacology ; Bacterial Proteins/genetics ; Ceftazidime/*pharmacology ; Cephalosporins/*pharmacology ; DNA, Bacterial/genetics/metabolism ; Drug Resistance, Bacterial/*drug effects ; Gram-Negative Bacteria/drug effects/*isolation & purification ; Humans ; Microbial Sensitivity Tests ; Penicillanic Acid/*analogs & derivatives/pharmacology ; Pseudomonas aeruginosa/drug effects/isolation & purification ; Real-Time Polymerase Chain Reaction

No abstract available.
Aged ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Cefotaxime/analogs & derivatives/therapeutic use ; Cholangiopancreatography, Endoscopic Retrograde ; Gallstones/surgery ; Gram-Negative Anaerobic Bacteria/drug effects/genetics/*isolation & purification ; Gram-Negative Bacterial Infections/*diagnosis/drug therapy/microbiology ; Humans ; Male ; Metronidazole/therapeutic use ; Microbial Sensitivity Tests ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; Sequence Analysis, DNA ; Tomography, X-Ray Computed

BACKGROUND: We evaluated the reliability and accuracy of the combined use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial identification and Vitek 2 antimicrobial susceptibility testing (AST) for bacteria from positive blood culture bottles. METHODS: Direct identification and AST were performed in parallel to the standard methods in monomicrobial positive blood culture bottles. In total, 254 isolates grown on aerobic and/or anaerobic bottles were identified with MALDI-TOF Vitek MS (bioMerieux, France), and 1,978 microorganism/antimicrobial agent combinations were assessed. For isolates from anaerobic bottles, an aliquot of the culture broth was centrifuged, washed, and filtered through a nylon mesh. For isolates from aerobic/pediatric bottles, a lysis step using 9.26% ammonium chloride solution and 2% saponin solution was included. RESULTS: The overall correct identification rate was 81.8% (208/254) and that for gram-positive/gram-negative isolates was 73.9%/92.6%, respectively, and it was 81.8%, 87.6%, and 57.9% for isolates from aerobic, anaerobic, and pediatric bottles, respectively. Identification was not possible in 45 cases, and most of these isolates were streptococci (N=14) and coagulase-negative staphylococci (N=11). Misidentification occurred only in one case. Compared with standard methods, direct AST showed 97.9% (1,936/1,978) agreement with very major error of 0.25%, major error of 0.05%, and minor error of 1.8%. CONCLUSIONS: This simple and cost-effective sample preparation method gives reliable results for the direct identification and AST of bacteria. For the identification of streptococci and coagulase-negative staphylococci, the method should be further improved.
Adult ; Ammonium Chloride/chemistry ; Anti-Infective Agents/*pharmacology ; Child ; Gram-Negative Bacteria/drug effects/*isolation & purification/metabolism ; Gram-Positive Bacteria/drug effects/*isolation & purification/metabolism ; Humans ; Reagent Kits, Diagnostic ; Saponins/chemistry ; *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

BACKGROUND: Rapid detection of carbapenemase-producing gram-negative bacilli (GNB) is required for optimal treatment of infected patients. We developed and assessed a new disk carbapenemase test (DCT). METHODS: Paper disks containing 0.3 mg of imipenem and bromothymol blue indicator were developed, and the performance of the DCT were evaluated by using 742 strains of GNB with or without carbapenemases. RESULTS: The paper disks were simple to prepare, and the dried disks were stable at -20℃ and at 4℃. The DCT detected 212 of 215 strains (98.6% sensitivity with 95% confidence interval [CI] 96.0-99.5%) of GNB with known class A (KPC and Sme) and class B (NDM, IMP, VIM, and SIM) carbapenemases within 60 min, but failed to detect GES-5 carbapenemase. The DCT also detected all two Escherichia coli isolates with OXA-48, but failed to detect GNB with OXA-232, and other OXA carbapenemases. The DCT showed 100% specificity (95% CI, 99.2-100%) in the test of 448 imipenem-nonsusceptible, but carbapenemase genes not tested, clinical isolates of GNB. CONCLUSIONS: The DCT is simple and can be easily performed, even in small laboratories, for the rapid detection of GNB with KPC, NDM and the majority of IMP, VIM, and SIM carbapenemases.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*metabolism ; Bromthymol Blue/chemistry ; Drug Resistance, Bacterial ; Gram-Negative Bacteria/drug effects/*enzymology ; Imipenem/pharmacology ; Microbial Sensitivity Tests/*methods ; Paper ; beta-Lactamases/*metabolism

OBJECTIVETo investigate the distribution of pathogenic bacteria in the patients with hematologic malignancies received hematopoietic stem cell transplantation (HSCT) and its influence on the expression of BCL-2 and BAX proteins.

METHODSThe clinical data of 64 patients with malignant lymphoma (ML) received auto-HSCT from January 2011 to December 2015 in our hospital were analyzed. On basis of post-treansplant infection, the patients were divided into infection group (36 cases) and non-infection group (28 cases). The distribution of pathogenic bacteria in 2 groups was identified, the T lymphocyte subsets of peripheral blood, expression level of apoptotic proteins and C-reaction protein (CRP) in 2 group were detected.

RESULTSThirty-six strains of pathogenic bacteria were isolated from 36 case of hematological malignancy after HSCT, including 24 strains of Gram-negative bacteria (66.67%) with predominamce of klebsiella pneumoniae (19.44%). The periperal blood CD4+ (t=2.637, P<0.01), CD4+/CD8+ ratio (t=8.223, P<0.01), BCL-2 protein (t=5.852, P<0.05), BCL-2/BAX ratio (t=14.56, P<0.01) in infection group were significantly lower than those in non-infection group, while CD8+ (t=2.285, P=<0.01), CRP (t=39.71, P<0.01), BAX level in infection group were higher than those in non-infection group. The pearson correcation analysis showed that the CD4+/CD8+ ratio in infection group positively correlated with BCL-2/BAX ratio (t=0.341, P<0.05), while serum CRP level in infection group negatively correlated with BCL-2/BAX ratio (t=-0.362, P<0.05).

CONCLUSIONThe pathogenic bacteria infecting ML patients after HSCT were mainly Gram-negative bacteria. The post-transplant infection can promote the expression up-regulation of related inflammatory factors and apoptotic proteins. The pathogens may be involved in cell apoptisis that provides a new strategy to treat the hematologic malignancies.

C-Reactive Protein ; analysis ; CD4-CD8 Ratio ; Gram-Negative Bacteria ; isolation & purification ; Hematologic Neoplasms ; metabolism ; microbiology ; Hematopoietic Stem Cell Transplantation ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; T-Lymphocyte Subsets ; cytology ; Up-Regulation ; bcl-2-Associated X Protein ; metabolism

Neonatal sepsis remains one of the most important causes of death and co-morbidity in very-low-birth-weight (VLBW) infants. The aim of this study was to determine the current incidences of early-onset sepsis (EOS) and late-onset sepsis (LOS), the distribution of pathogens, and the impact of infection on co-morbidities in VLBW infants. We analyzed the data including sepsis episode from 2,386 VLBW infants enrolled in Korean Neonatal Network from January 2013 to June 2014. We defined EOS as a positive blood culture occurring between birth and 7 days of life and LOS after 7 days of life. Sepsis was found in 21.1% of VLBW infants. The risk of sepsis was inversely related to birth weight and gestational age. EOS was found in only 3.6% of VLBW infants, however the mortality rate was as high as 34.1%. EOS was associated with the increased odds for bronchopulmonary dysplasia and intraventricular hemorrhage. The vast majority of EOS was caused by Gram-positive organisms, particularly coagulase-negative staphylococci (30.6%). LOS developed in 19.4% of VLBW infants with a 16.1% mortality rate. Pathogens in LOS were dominated by coagulase-negative staphylococci (38.3%). Twenty-five percent and fifty percent of first LOS episode occurred after 12 days and 20 days from birth, respectively. Younger and smaller VLBW infants showed the earlier occurrence day for the 25% of first LOS episode. This study provides a recent nationwide epidemiology of sepsis in VLBW infants in Korea. Based on this study, successful strategies to reduce infections would improve survival and reduce morbidity.
Coagulase/metabolism ; Databases, Factual ; Gestational Age ; Gram-Negative Bacteria/isolation & purification ; Gram-Positive Bacteria/isolation & purification ; Humans ; Incidence ; Infant, Newborn ; *Infant, Very Low Birth Weight ; Kaplan-Meier Estimate ; Republic of Korea/epidemiology ; Risk Factors ; Sepsis/*epidemiology/microbiology/mortality ; Staphylococcus/enzymology/isolation & purification