Rapid and precise identification of bloodstream infections using a pre-treatment protocol combined with high-throughput multiplex genetic detection system.

BACKGROUND: Bloodstream infection (BSI) is a life-threatening condition with high morbidity and mortality rates worldwide. Early diagnosis of BSI is critical to avoid the unnecessary application of antimicrobial agents and for proper treatment. However, the current standard methods based on blood culture are time-consuming, thus failing to provide a timely etiological diagnosis of BSI, and common PCR-based detection might be inhibited by matrix components. METHODS: The current study explored an integrated pre-analytical treatment protocol for whole blood samples, wherein pathogens are enriched and purified by incubation and concentration, and inhibitors are inactivated and removed. Further, this study developed and evaluated a novel high-throughput multiplex genetic detection system (HMGS) to detect 24 of the most clinically prevalent BSI pathogens in blood culture samples and pre-treated whole blood samples. The specificity and sensitivity were evaluated using related reference strains and quantified bacterial/fungal suspensions. The clinical utility of BSI-HMGS combined with the pre-analytical treatment protocol was verified using blood cultures and whole blood samples. RESULTS: The combined pre-treatment protocol and BSI-HMGS was highly specific for target pathogens and possessed a low detection limit for clinical whole blood samples. The pre-treatment protocol could deplete the PCR inhibitors effectively. For blood culture samples, the current method showed 100.0% negative percent agreements and > 87.5% positive percent agreements compared to the reference results based on blood culture findings. For whole blood samples, the current method showed 100.0% negative percent agreements and > 80.0% positive percent agreements compared to the reference results for most pathogens. The turnaround time was ≤ 8 h, and all the procedures could be conducted in a general clinical laboratory. CONCLUSION: The BSI-HMGS combined with the pre-treatment protocol was a practical and promising method for early and precise detection of BSIs, especially for areas without access to advanced medical facilities.

Developing a multiplex PCR-based assay kit for bloodstream infection by analyzing genomic big data.

BACKGROUND: In recent years, the incidence of bloodstream infections (BSI) has increased, the composition of pathogenic bacteria has changed, and drug resistance among bacteria has gradually increased due to the widespread use of interventional techniques, broad-spectrum antibacterial drugs, hormones, and immunosuppressive agents. Here, we have developed a multiplex PCR assay kit for the detection of pathogens (14 Gram-negative bacteria, 15 Gram-positive bacteria, and 4 fungi) in whole blood from patients with BSI using five-color fluorescent multiplex PCR followed by capillary electrophoresis. Our assay exhibits a diagnosis of higher quality and an improved detection rate for common pathogens. METHODS: A local genome DNA database of 33 pathogenic bacteria was constructed. Next, "Exhaustive" primer search of the full coding sequence of the reference genomes of these bacteria was performed. Panels with minimal interactions between primers and amplicons were selected by random sampling and testing by a recursive algorithm. Primers and Mg2+ concentrations and PCR reaction procedures were optimized to maximize the detection efficacy. RESULTS: The LOD of the kit was determined as 100 copies/µl. Using clinical samples, results generated by this kit and regular blood culture method were found to be 95.08% consistent. Additionally, six pathogens which were unidentifiable by blood culture were successfully detected by this kit. CONCLUSION: Our study provided a bioinformatics approach to the challenge of primer design in multiplex PCR, and combined with optimized wet lab practice, a multiplex PCR-based assay kit for BSI with higher sensitivity and accuracy than blood culture was produced.

Simultaneous detection of 9 respiratory pathogens using a newly developed multiplex real-time PCR panel based on an automatic molecular detection and analysis system.

Timely identification of respiratory pathogens guides specific treatment, reduces hospital costs and minimizes the excessive use of antibiotics. A new multiplex real-time PCR panel was developed based on an automatic molecular detection and analysis system (AutoMolec system), consisting of three separate internally controlled assays. Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, human metapneumovirus, influenza B virus, respiratory syncytial virus and human parainfluenza virus 1-3 may be directly detected in original samples. The system's clinical performance was evaluated by comparison with an approved commercial kit, using 517 clinical samples. The limit of detection of the AutoMolec mRT-PCR panel ranged from 4 × 10-4 ∼3.3 TCID50/mL and no cross-reaction with common respiratory pathogens was observed. The AutoMolec mRT-PCR panel had 99.09% sensitivity and 100.0% specificity and overall detection consistency was 99.61%, making it comparable to that of the commercial kit. Therefore, the AutoMolec mRT-PCR panel has great potential for routine screening of respiratory infection in China.

Establishment and evaluation of a quadruple quantitative real-time PCR assay for simultaneous detection of human coronavirus subtypes.

BACKGROUND: The newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four seasonal human coronaviruses (HCoVs) (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1) still circulate worldwide. The early clinical symptoms of SARS-CoV-2 and seasonal HCoV infections are similar, so rapid and accurate identification of the subtypes of HCoVs is crucial for early diagnosis, early treatment, prevention and control of these infections. However, current multiplex molecular diagnostic techniques for HCoV subtypes including SARS-CoV-2 are limited. METHODS: We designed primers and probes specific for the S and N genes of SARS-CoV-2, the N gene of severe acute respiratory syndrome coronavirus (SARS-CoV), and the ORF1ab gene of four seasonal HCoVs, as well as the human B2M gene product. We developed and optimized a quadruple quantitative real-time PCR assay (qq-PCR) for simultaneous detection of SARS-CoV-2, SARS-CoV and four seasonal HCoVs. This assay was further tested for specificity and sensitivity, and validated using 184 clinical samples. RESULTS: The limit of detection of the qq-PCR assay was in the range 2.5 × 101 to 6.5 × 101 copies/µL for each gene and no cross-reactivity with other common respiratory viruses was observed. The intra-assay and inter-assay coefficients of variation were 0.5-2%. The qq-PCR assay had a 91.9% sensitivity and 100.0% specificity for SARS-CoV-2 and a 95.7% sensitivity and 100% specificity for seasonal HCoVs, using the approved commercial kits as the reference. Compared to the commercial kits, total detection consistency was 98.4% (181/184) for SARS-CoV-2 and 98.6% (142/144) for seasonal HCoVs. CONCLUSION: With the advantages of sensitivity, specificity, rapid detection, cost-effectiveness, and convenience, this qq-PCR assay has potential for clinical use for rapid discrimination between SARS-CoV-2, SARS-CoV and seasonal HCoVs.

A New Multiplex Genetic Detection Assay Method for the Rapid Semi-Quantitative Detection of Six Common Curable Sexually Transmitted Pathogens From the Genital Tract.

Background: Sexually transmitted infections (STIs) are some of the most common communicable conditions and exert impact on the health and lives of many hundreds of millions of people across the world every year. Screening high-risk populations and conducting comprehensive detection tests would lead to a significant improvement in preventing the transmission of STIs and help us to provide rapid treatment to those affected. Here, we successfully established and validated a novel high-throughput multiplex gene detection system (HMGS) for the simultaneous and semiquantitative detection of six important curable sexually transmitted pathogens in a single reaction from secretions samples. Method: Fluorescently labeled primers were designed to target specific conserved and single-copy gene fragments of Ureaplasma urealyticum (U. urealyticum), Mycoplasma hominis (M. hominis), Chlamydia trachomatis (C. trachomatis), Neisseria gonorrhoeae (N. gonorrhoeae), Trichomonas vaginalis (T. vaginalis), and Treponema pallidum (T. pallidum). The specificity and sensitivity of the STI-HMGS was validated and optimized using plasmids and quantitative genomic DNA. Next, we validated the performances of the STI-HMGS for clinical application by testing samples of clinical secretions collected from patients who visited the gynecology and urology outpatient clinics of our reproductive medicine center. Results derived from the STI-HMGS were then compared with three approved commercialized kits that used to detect U. urealyticum, C. trachomatis and N. gonorrhoeae, respectively, followed by further validation with Sanger sequencing for all pathogens. Finally, a comprehensive analysis of epidemiology was performed among different subgroups to investigate the association between infection rates and clinically-relevant information. Results: The sensitivity of STI-HMGS for six target genes was 10 copies/µL. Data derived from the detection of 381 clinical secretions demonstrated that the STI-HMGS exhibited high concordance rate compared with approved commercialized kits and almost 100% sensitivity and specificity for the detection of six sexually transmitted pathogens when validated by Sanger sequencing. Semi-quantitative analysis found that STIs caused by N. gonorrhoeae had a significantly higher (P<0.05) pathogen load than the other pathogens. Infections caused by C. trachomatis were significantly more common in younger individuals (P<0.05). We also found that U. urealyticum infections were more likely to happen in females; while the males were more affected by N. gonorrhoeae (P<0.05). Conclusions: STI-HMGS proved to be an efficient method for the semi-quantitative detection of six important curable sexually transmitted pathogens and therefore represents an alternative method for the clinical detection and monitoring of STIs.

iTRAQ-Based Quantitative Proteomic Profiling of Staphylococcus aureus Under Different Osmotic Stress Conditions.

Staphylococcus aureus (S. aureus) is an extremely halotolerant pathogenic bacterium with high osmotic stress tolerance, and it is frequently encountered in aquatic production and preservation. However, the mechanism underlying the extremely high osmotic stress tolerance of S. aureus remains unclear. In this study, the isobaric tags for relative and absolute quantification (iTRAQ) method was used to identify the differentially expressed proteins (DEPs) under different sodium chloride (NaCl) concentrations. Compared with the control group (0% NaCl), the 10 and 20% NaCl groups had 484 DEPs and 750 DEPs, respectively. Compared with the 10% NaCl group, the 20% NaCl group had 361 DEPs. Among the DEPs, proteins involved in fatty acid synthesis, proline/glycine betaine biosynthesis and transportation, stress tolerance, cell wall biosynthesis and the TCA cycle were upregulated, whereas proteins associated with biofilm formation and pathogenic infections were downregulated. The results obtained in this study indicate that under extremely high osmotic stress, modification of the cell membrane structure, increased biosynthesis and transportation of osmotic protectants, and redistribution of energy metabolism contribute to the osmotic stress tolerance of S. aureus, and the infectious ability of the bacteria may be limited. The aim of this study was to provide new insight into how S. aureus tolerates the high-salt conditions involved in aquatic production and preservation.

Sex-Based Differences in Gut Microbiota Composition in Response to Tuna Oil and Algae Oil Supplementation in a D-galactose-Induced Aging Mouse Model.

Our previous work indicated that a mixture of tuna oil and algae oil treatment in male mice effectively relieved D-galactose (D-gal)-induced aging and resulted in gut microbiota alterations, and that the best anti-aging effects were observed for a tuna oil to algae oil ratio of 1:2. However, the possibility of a sex-based difference in the anti-aging effect of the tuna oil and algae oil mixture or gut microbiota variation, has rarely been investigated. In this study, the anti-aging effect of an oil mixture (1:2) in male and female mice was measured, and oil treatment improved the learning and cognition of mice that were damaged by D-gal, increased the activities of anti-oxidative enzymes, and decreased the level of MDA, which acted as a hallmark of oxidative damage to lipids. Male mice showed better anti-aging effects than female mice with a specific oil mixture ratio, and the clinical drug donepezil showed a similar or better effect on aging alleviation than oil treatments in both sexes. On the other hand, the same oil treatment led to different gut microbiota composition alterations in male and female mice. Redundancy analysis (RDA) identified 31 and 30 key operational taxonomic units (OTUs) in the male and female mice, respectively, and only three of these OTUs overlapped. Moreover, the abundance of Lactobacillus and several probiotic-like butyric acid producers was higher in male mice than in female mice, whereas the abundance of some inflammation-related genera, such as Clostridium XlVa, was lower in male mice. In conclusion, this study indicated the sex-based differences related to the anti-aging effects of tuna oil and algae oil treatment are accompanied by sex-based differences in gut microbiota modulation.

Tuna Oil Alleviates d-Galactose Induced Aging in Mice Accompanied by Modulating Gut Microbiota and Brain Protein Expression.

To discern whether tuna oil modulates the expression of brain proteins and the gut microbiota structure during aging induced by d-galactose, we generated an aging mouse model with d-galactose treatment, and the mice showed aging and memory deterioration symptoms according to physiological and biochemical indices. Treatment with different doses of tuna oil alleviated the symptoms; the high dose showed a better effect. Subsequently, brain proteomic analysis showed the differentially expressed proteins were involved in damaged synaptic system repairment and signal transduction system enhancement. In addition, tuna oil treatment restored the diversity of gut microbiota, 27 key operational taxonomic units, which were identified using a redundancy analysis and were significantly correlated with at least one physiological index and three proteins or genes. These findings suggest that the combination of proteomics and gut microbiota is an effective strategy to gain novel insights regarding the effect of tuna oil treatment on the microbiota-gut-brain axis.

Modulation of gut microbiota by dietary supplementation with tuna oil and algae oil alleviates the effects of D-galactose-induced ageing.

Previous studies have shown that dietary supplementation with tuna oil and algae oil can alleviate the effects of ageing on learning and memory in mouse models, but the mechanism of this effect remains unknown. This study aimed to determine whether dietary oil supplementation alters the composition of the gut microbiota during the prevention of age-related effects on cognition. Ageing mice received dietary oil supplementation continuously for 12 weeks. The supplementation was found to improve the animals' learning and cognition, and this effect was most marked in the TO200AO400 group, which received a 1:2 mixture of tuna oil and algae oil at 600 mg kg-1 day-1. Next-generation sequencing of the 16S rRNA gene present in faecal samples showed that the gut microbiota varied in the groups that received different oil treatments; the TO200AO400 treatment most closely restored the composition of the D-galactose-altered gut microbiota to that of the control. Moreover, 83 altered operational taxonomic units (OTUs) responsive to dietary oil supplementation were identified; five of these differed in one or more parameters associated with host ageing. In conclusion, this study confirmed the effect of dietary oil supplementation on the alleviation of age-related decline in cognitive function and showed that oil supplementation results in alterations in the composition of the gut microbiota. Further research will be needed to elucidate the causal relationship between the reversal of age-related cognitive decline and gut microbiota modulation and to explore the potential of gut microbial communities as a diagnostic biomarker and a therapeutic target in ageing.

Dietary Apostichopus japonicus Alleviates Diabetes Symptoms and Modulates Genes Expression in Kidney Tissues of db/db Mice.

The effects of Apostichopus japonicus enzymatic hydrolysate on the regulation of dyslipidemia, pathoglycemia, and transcription changes in kidney tissues of db/db mice were evaluated. In this study, the symptoms of diabetes in db/db mice were alleviated after 10 weeks of treatments with low (db/db + LD group) and high dose (db/db + HD group) of Apostichopus japonicus enzymatic hydrolysate, and the high dose treatment showed a better antidiabetic effect. Compared with the db/db group, the fasting blood glucose levels (36.84 ± 7.82 vs 25.18 ± 6.84 mmol/L, P < 0.01), the urine glucose levels (45.44 ± 3.93 vs 22.66 ± 5.58 mmol/L, P < 0.01), and the serum insulin sensitivity index (-4.65 ± 0.43 vs -4.74 ± 0.75, P > 0.05) in the db/db + HD group were decreased, whereas the fasting plasma insulin (3.12 ± 1.08 vs 5.54 ± 1.82 µg/L, P < 0.01) and the serum insulin resistance index (5.01 ± 2.02 vs 5.96 ± 2.49, P < 0.05) were increased. Subsequently, the kidney transcription profiles were measured in the db/db group and db/db + HD group via microarray, and the results show that Apostichopus japonicus hydrolysate induced differential expression of 77 genes. Among these genes, the down-regulation of genes ntrK1 and ptpN5 played vital roles, as this effect induced the further down-regulation of neurotrophin tyrosine kinase, protein tyrosine phosphatase, and other transcription factors, which are involved in the classical mitogen-activated protein kinases (MAPK) and p38MAPK signaling pathways. The inhibited MAPK and p38MAPK signaling pathways are involved in glycometabolism and the control of lipid metabolism, and they regulate the occurrence and development of diabetic nephropathy.

Microbial diversity and composition in different gut locations of hyperlipidemic mice receiving krill oil.

Low-dose (LD, 100 mg kg-1 day-1), moderate-dose (MD, 200 mg kg-1 day-1), and high-dose (HD, 600 mg kg-1 day-1) krill oil treatments have a stepwise, enhanced effect on alleviating hyperlipidemia, and 16S rRNA sequencing of the fecal samples demonstrates that krill oil treatment alters microbial communities. Feces may not represent all microbial communities in the gastrointestinal (GI) tract. Therefore, in this study, the stored ileal and colon samples collected from LD and HD groups were sequenced, and the location-specific modulations of microbial communities were observed after krill oil treatments. The 16S rRNA sequencing of the ileal samples showed that the LD and HD groups have similar patterns between control and high-fat diet (HFD) treatments, and six most abundant genera and 40 operational taxonomic units that respond to krill oil treatment were identified. However, the 16S rRNA sequencing of the colon samples showed that LD krill oil shifts the structure from the HFD to that of the control, whereas the HD group was distributed between the control and HFD groups. The corresponding most abundant genera and responsive OTUs totaled 4 and 45, respectively. In conclusion, different gastrointestinal tract locations contain different microbial communities. These results will help to provide a comprehensive understanding of the role of dietary krill oil in modulating the gut microbiota and alleviating hyperlipidemia.

Modulation of the gut microbiota by the mixture of fish oil and krill oil in high-fat diet-induced obesity mice.

Previous studies confirmed that dietary supplements of fish oil and krill oil can alleviate obesity in mice, but the underlying mechanism remains unclear. This study aims to discern whether oil treatment change the structure of the gut microbiota during the obesity alleviation. The ICR mice received high-fat diet (HFD) continuously for 12 weeks after two weeks of acclimatization with a standard chow diet, and the mice fed with a standard chow diet were used as the control. In the groups that received HFD with oil supplementation, the weight gains were attenuated and the liver index, total cholesterol, triglyceride and low-density lipoprotein cholesterol were reduced stepwise compared with the HFD group, and the overall structure of the gut microbiota, which was modulated in the HFD group, was shifted toward the structure found in the control group. Moreover, eighty-two altered operational taxonomic units responsive to oil treatment were identified and nineteen of them differing in one or more parameters associated with obesity. In conclusion, this study confirmed the effect of oil treatment on obesity alleviation, as well as on the microbiota structure alterations. We proposed that further researches are needed to elucidate the causal relationship between obesity alleviation and gut microbiota modulation.

Proteomics and 1H NMR-based metabolomics analysis of pathogenic Vibrio vulnificus aquacultures isolated from sewage drains.

Vibrio bacteria live in both marine and freshwater habitats and are associated with aquatic animals. Vibrio vulnificus is a pathogenic bacterium that infects people and livestock. It is usually found in offshore waters or within fish and shellfish. This study presents a comparative proteomic analysis of the outer membrane protein (OMP) changes in V. vulnificus proteins after stimulation with sewage from sewage drains. Using two-dimensional electrophoresis followed by MALDI-TOF MS/MS, 32 protein spots with significant differences in abundance were identified and characterized. These identified proteins were found to be involved in various functional categories, including catalysis, transport, membrane proteins progresses, receptor activity, energy metabolism, cytokine activity, and protein metabolism. The mRNA expression levels of 12 differential proteins were further assessed by qRT-PCR. Seven genes including carboxypeptidase, hemoglobin receptor, succinate dehydrogenase iron-sulfur subunit, ATP synthase subunit alpha, thioredoxin, succinyl-CoA synthetase subunit, and alanine dehydrogenase were downregulated upon stimulation, whereas the protein expression levels HupA receptor, type I secretion outer membrane protein, glutamine synthetase, superoxide dismutase, OmpU, and VuuA were upregulated. 1H NMR spectra showed 18 dysregulated metabolites from V. vulnificus after the sewage stimulation and the pathogenicity was enhanced after that.

NMR-based metabolomics reveals the metabolite profiles of Vibrio parahaemolyticus under ferric iron stimulation.

Vibrio parahaemolyticus is a halophilic bacterium endemic to coastal areas, and its pathogenicity has caused widespread seafood poisoning. In our previous research, the protein expression of V. parahaemolyticus in Fe3+ medium was determined using isobaric tags for relative and absolute quantitation (iTRAQ). Here, nuclear magnetic resonance (NMR) was used to detect changes in the V. parahaemolyticus metabolome. NMR spectra were obtained using methanol-water extracts of intracellular metabolites from V. parahaemolyticus under various culture conditions, and 62 metabolites were identified, including serine, arginine, alanine, ornithine, tryptophan, glutamine, malate, NAD+, NADP+, oxypurinol, xanthosine, dCTP, uracil, thymine, hypoxanthine, and betaine. Among these, 21 metabolites were up-regulated after the stimulation of the cells by ferric iron, and 9 metabolites were down-regulated. These metabolites are involved in amino acid and protein synthesis, energy metabolism, DNA and RNA synthesis and osmolality. Based on these results, we conclude that Fe3+ influences the metabolite profiles of V. parahaemolyticus.

Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta.

Ferritins are primary iron storage proteins and play a crucial role in iron storage and detoxification. Yeast two-hybrid method was employed to screen the cDNA library of Phascolosoma esculenta. Sequence of positive colony FER147 was analyzed. The higher similarity and conserved motifs for ferritin indicated that it belonged to a new member of ferritin family. The interaction between Ferritin and Fer147 was further confirmed through co-immunoprecipitation. The pET-28a-FER147 prokaryotic expression vector was constructed. The expressed recombinant Fer147 was then isolated, purified, and refolded. When ferritins were treated by different heavy metals, several detection methods, including scanning electron microscopy (SEM), circular dichroism (CD), and inductively coupled plasma-mass spectrometry (ICP-MS) were applied to examine the structures and functions of the new protein Fer147, recombinant P. esculenta ferritin (Rferritin), and natural horse-spleen ferritin (Hferritin). SEM revealed that the three ferritin aggregates changed obviously after different heavy metals treatment, meanwhile, a little different in aggregates were detected when the ferritins were trapped by the same heavy metal. Hence, changes in aggregation structure of the three proteins are related to the nature of the different heavy metals and the interaction between the heavy metals and the three ferritins. CD data suggested that the secondary structure of the three ferritins hardly changed after different heavy metals were trapped. ICP-MS revealed that the ferritins exhibit different enrichment capacities for various heavy metals. In particular, the enrichment capacity of the recombinant Fer147 and Rferritin is much higher than that of hferritin.

Modulation of the Gut Microbiota by Krill Oil in Mice Fed a High-Sugar High-Fat Diet.

Multiple lines of evidence suggest that the gut microbiota plays vital roles in metabolic diseases such as hyperlipidemia. Previous studies have confirmed that krill oil can alleviate hyperlipidemia, but the underlying mechanism remains unclear. To discern whether krill oil changes the structure of the gut microbiota during the hyperlipidemia treatment, 72 mice were acclimatized with a standard chow diet for 2 weeks and then randomly allocated to receive a standard chow diet (control group, n = 12) or a high-sugar-high-fat (HSHF) diet supplemented with a low (100 µg/g·d, HSHF+LD group, n = 12), moderate (200 µg/g·d, HSHF+MD group, n = 12) or high dosage of krill oil (600 µg/g·d, HSHF+HD group, n = 12), simvastatin (HSHF+S group, n = 12) or saline (HSHF group, n = 12) continuously for 12 weeks. The resulting weight gains were attenuated, the liver index and the low-density lipoprotein, total cholesterol and triglyceride concentrations showed a stepwise reduction in the treated groups compared with those of the control group. A dose-dependent modulation of the gut microbiota was observed after treatment with krill oil. Low- and moderate- doses of krill oil increased the similarity between the composition of the HSHF diet-induced gut microbiota and that of the control, whereas the mice fed the high-dose exhibited a unique gut microbiota structure that was different from that of the control and HSHF groups. Sixty-five key operational taxonomic units (OTUs) that responded to the krill oil treatment were identified using redundancy analysis, of which 26 OTUs were increased and 39 OTUs were decreased compared with those of the HSHF group. In conclusion, the results obtained in this study suggest that the structural alterations in the gut microbiota induced by krill oil treatment were dose-dependent and associated with the alleviation of hyperlipidemia. Additionally, the high-dose krill oil treatment showed combined effects on the alleviation of hyperlipidemia and obesity.

A metabonomic analysis on the response of Enterobacter cloacae from coastal outfall for land-based pollutant under phoxim stress.

Enterobacter cloacae is an opportunistic pathogen widely distributed in human and animal intestinal systems. The secretion of extended-spectrum ß-lactamases (ESBLs) and cephalosporinase (AmpC) endows E. cloacae with strong drug resistance. In a previous study by our group, protein expression of E. cloacae under phoxim stress was measured by two-dimensional electrophoresis. Here, nuclear magnetic resonance was used to detect differences in E. cloacae metabonomics when under phoxim stress. We determined that there are 29 types of metabolites that differ between phoxim stress and normal culture conditions. Among these, 6 types of metabolites were upregulated in the phoxim stress group, and 23 types of metabolites were inhibited. Though enrichment analysis, seven pathways were identified by different expression levels of metabolites, which were involved in DNA and RNA synthesis, DNA damage repair, antioxidation and functions of the cell membrane and cell wall. The mechanism underlying how phoxim affects E. cloacae was determined by studying the results of both two-dimensional electrophoresis in our prior work and the analysis of E. cloacae metabonomic changes under phoxim stress.

Structural modulation of gut microbiota in Bama minipigs in response to treatment with a "growth-promoting agent", salbutamol.

Even though salbutamol (SAL) had remarkable effects on the enhancement of growth rate and carcass composition in different livestock species such as cattle, pigs, sheep and poultry, it was banned as a growth promoter because of its adverse effects on health. However, the specific mechanism by which salbutamol enhances growth efficiency remains unknown. In this study, Bama pigs were randomly allocated to receive salbutamol (5 mg/kg) for 30 or 60 days and were compared with untreated pigs. Pigs treated with salbutamol demonstrated enhanced growth rates and carcass composition; however, they showed deterioration in blood biochemical indices and organ development. We hypothesized that salbutamol exerts its effects by modulating the composition of the gut microbiota population. The faecal microbiome of pigs was characterized via pyrosequencing of the bacterial 16S rRNA gene. The gut microbiota population analysis showed that salbutamol caused shifts in the microbial composition of less abundant species. Redundancy analysis indicated an increase in abundance of the phylum Bacteroidetes, class Betaproteobacteria, family Christensenellaceae and genus Lactobacillus, and a decreased ratio of the phylum Firmicutes, class Clostridia and genera Ruminococcus, Blautia and Subdoligranulum. In conclusion, our study provided circumstantial evidence that the various effects of salbutamol are caused by gut microbiota modulation, and several potential candidates were identified for SAL detection via the gut microbiota. Our findings provided new insights into the roles of the gut microbiota during salbutamol treatment, and these findings will aid in the screening of alternative strategies for animal health improvement and production enhancement.

A Comparison of Molecular Biology Mechanism of Shewanella putrefaciens between Fresh and Terrestrial Sewage Wastewater.

Municipal and industrial wastewater is often discharged into the environment without appropriate treatment, especially in developing countries. As a result, many rivers and oceans are contaminated. It is urgent to control and administer treatments to these contaminated rivers and oceans. However, most mechanisms of bacterial colonization in contaminated rivers and oceans were unknown, especially in sewage outlets. We found Shewanella putrefaciens to be the primary bacteria in the terrestrial sewage wastewater outlets around Ningbo City, China. Therefore, in this study, we applied a combination of differential proteomics, metabolomics, and real-time fluorescent quantitative PCR techniques to identify bacteria intracellular metabolites. We found S. putrefaciens had 12 different proteins differentially expressed in freshwater culture than when grown in wastewater, referring to the formation of biological membranes (Omp35, OmpW), energy metabolism (SOD, deoxyribose-phosphate pyrophosphokinase), fatty acid metabolism (beta-ketoacyl synthase), secondary metabolism, TCA cycle, lysine degradation (2-oxoglutarate reductase), and propionic acid metabolism (succinyl coenzyme A synthetase). The sequences of these 12 differentially expressed proteins were aligned with sequences downloaded from NCBI. There are also 27 differentially concentrated metabolites detected by NMR, including alcohols (ethanol, isopropanol), amines (dimethylamine, ethanolamine), amino acids (alanine, leucine), amine compounds (bilinerurine), nucleic acid compounds (nucleosides, inosines), and organic acids (formate, acetate). Formate and ethanolamine show significant difference between the two environments and are possibly involved in energy metabolism, glycerophospholipid and ether lipids metabolism to provide energy supply, and material basis for engraftment in sewage. Because understanding S. putrefaciens's biological mechanism of colonization (protein, gene express, and metabolites) in terrestrial sewage outlets is so important to administering and improving contaminated river and to predicting and steering performance, we delved into the biological mechanism that sheds light on the effect of environmental conditions on metabolic pathways.

Multi-omics analysis on the pathogenicity of Enterobacter cloacae ENHKU01 isolated from sewage outfalls along the Ningbo coastline.

BACKGROUND: The acquisition of iron is important for the pathogenicity of bacteria and blood. Three different culture environments (Fe stimulation, blood agar plate and normal plate) were used to stimulate Enterobacter cloacae, and their respective pathogenicities were compared at the proteomic, mRNA and metabolomic levels. METHODS: 2D-DIGE combined with MALDI-TOF-MS/MS, RT-PCR and 1H NMR were used to analyze the differential expression levels of proteins, mRNA and metabolites. RESULTS: A total of 109 proteins were identified by 2D-DIGE and mass spectrometry after pairwise comparison within three culture environments, clustered into 3 classes and 183 functional categories, which were involved in 23 pathways. Based on the 2D-DIGE results, multiple proteins were selected for verification by mRNA expression. These results confirmed that most of the proteins were regulated at the transcriptional level. Thirty-eight metabolites were detected by NMR, which correlated with the differentially expressed proteins under different treatment conditions. CONCLUSIONS: The results show that culture in a blood agar plate and a suitable concentration of iron promote the pathogenicity of E. cloacae and that high iron concentrations may have adverse effects on growth and iron uptake and utilization by E. cloacae.