| Acinetobacter baumannii is now recognized as one of the most successful opportunistic pathogens for the global health care institutions, while it was previously considered as a low-virulence bacterium. Although many Acinetobacter species are ubiquitous and easily recovered from many natual environments, there is no known natural habitat for A. baumannii outside the hospital. With the identified multifarious virulence factors, A. baumannii has been implicated in a variety of nosocomial infections associated with high morbidity or mortality rates, including ventilator-associated pneumonia, wound infections, bloodstream infections, secondary meningitis, skin and soft tissue infections and urinary tract infections et al. The formation of biofilms on both biotic and abiotic surfaces and resistance to multiple antibacterial agents seem to play important roles in the pathogenicity of A. baumannii.Many A. baumannii clinical strains are capable of attaching their planktonic cells to different hydrophilic or hydrophobic substrata, e.g. hospital equipment, indwelling medical devices and bronchial epithelial cells, and growing as biofilms at the solid-liquid interface. As the self-produced extracellular matrix (ECM) enclosed microbial accretions that adhere to surfaces, and the formed bioflm on surfaces. Biofilms offer A. baumannii a stable and protected lifestyle, and allow it to survive in hostile environments and disperse to colonize new niches, which results in a higher risk of nosocomial infections.To understand the effect of biofilm formation on different properties of the substratum surface, we estimated the ability of A. baumannii ATCC 19606 type strain, and one colistin-resistant variant of A. baumannii ATCC 19606 to form biofilm on four different substratum surfaces. Four different surfaces including hydrophilic plastic, hydrophobic plastic, hydrophilic glass, and hydrophobic glass were tested. The results showed that both A. baumannii ATCC 19606 wild type strain and colistin-resistant mutant strain tended to form more biofilm on hydrophobic surfaces compared with hydrophilic surfaces, whether on plastic or glass. At the same time we also found that SDS and ultrasonic cleaning process could effectively remove residues of biofilm. As for the biofilm formed by A. baumannii, the effect of SDS processing might be better than that of the ultrasonic cleaning process.Besides the formation of biofilm on different surfaces, the predominance of reported clinical A. baumannii isolates has been related to the multidrug-resistant (MDR) phenotype, which is a consequence of a variety of resistance mechanisms against different antibiotics, i.e. permeability defects, expression of multidrug efflux pumps, production of antibiotic-degradation/modification enzymes and alteration of drug-targeting sites. According to the recent genomic analyses, the high capacity to acquire new genetic determinants contributes A. baumannii to display an open pan genome, which may act a key role in the development of MDR phenotype. Unlike A. baumannii, Myxococcus xanthus is ubiquitous, soil-dwelling, Gram-negative non-pathogen bacteria. M. xanthus is a predatory δ-proteobacterium that is able to grow by lysing a variety of prey bacteria and metabolizing the nutrients released, and it produces myxovirescin (antibiotic TA) antibiotics functioning as a weapon in the predation. A. baumaimii is an opportunistic Gram-negative bacterial pathogen, which is associated with a range of nosocomial infections. The treatment of these infections is hampered by the rapid rise in prevalence of A. baumaimii strains that are resistant to almost all available antibiotics, and colistin (polymyxin E) is often the only remaining treatment, although colistin-resistant clinical isolates have already been reported. In a preliminary screening, we found that M. xanthus DK1622 cells could prey on the colistin-resistant A. baumannil isolates but not on the colistin-susceptible A. baumaimii wild-type cells. Here, we sought to elucidate resistant mechanism for A. baumannii to M. xanthus predation. We showed that the A. baumannii wild-type cells were resistant to the antibiotic TA and colistin-resistant variants were susceptible to TA treatment, while the lspA genes and their transcriptions remained the same in both of these two kind of cells, which encode type II signal peptidase LspA as the TA’s target. It has been demonstrated that A. baumannii develop resistance to colistin by complete loss of lipopolysaccharide (LPS) production. After dispelling the concern that A. baumaimii LPS might directly interact and titrate TA, we showed that missing of LPS in colistin-resistant A. baumannii strain compromised out membrane of cells and led to their susceptibility to TA treatment. The normal outer membrane of A. baumannii wild-type cells might provide a formidable barrier for penetration of TA, which resulted in their resistance to TA and M. xanthus predation. Our study suggests that combination of antibiotics targeting different cellular processes may be an effective strategy to treat the infections caused by multidrug-resistant bacteria.The strong aromatic Chinese liquors are fermented by Daqu with a variety of mixed microorganisms in natural environments. A variety of microorganisms, growing in the pit mud participate in the brewing process, and their metabolites constitute the main flavor components of the liquor. In the liquor brewing industry, it is a mystery that older cellars produce better tasting liquors. The strong aromatic Chinese liquors are batch-brewed in mud cellars, but the changes in the brewing microflora and their effects on the production of flavors are mostly unclear.In order to investigat the community structures of microorganism in the cellar mud. We selected two neighbored cellars, a 1-year-old cellar and a 10-year-old cellar, which have not been improved during the brewing periods, to analyze the effects of long-time batch brewing on the microflora in the cellar mud. We shoveled the mud samples from the top site (0-20 cm bellow the upper edge), the middle site (80-100 cm below the upper edge) and the bottom of cellar. After collection, the genomic DNA was extracted from the cellar mud. A set of primers was designed to amplify the V3-V5 hypervariable region (approximately 500 bp) of the 16S rRNA gene to construct libraries containing the 16S rRNA gene sequences of bacteria and archaea. The entire pyrosequencing data set from the six samples contained 60,894 sequences. After removing the barcode and primer sequences, we further removed the sequences contained any chimera sequences, ambiguous bases or with homopolymers longer than 8 bp using the mothur software package (version 1.27.0, http://www.mothur.org/). After filtering, 54,634 high-quality sequences (89.7%of the total sequences) remained, with an average read length of 514±60 bp.The microbial communities in the six samples could be classified into two groups:the 10-year-old cellar samples and the 1-year-old cellar samples. The map showed that the bacteria in the old cellar mud were not only significantly different in composition from, but also much richer than those in the new cellar mud, especially Archaea. The results also showed that after a decade of brewing acclimation, the distribution differences of bacteria between the layers of cellar were significantly reduced.Microorganisms in the cellar mud involved in the whole fermentation process, and their metabolic behavior also affected the quality of liquor. To estimate the production abilities of volatile compounds by different cellar bacteria, we isolated 32 different bacterial strains from cellar mud samples. After phylogenetic analysis, we selected ten strains and cultivated them to assay their production abilities of low-molecular-weight organic compounds. These ten representatives belonged to the genera of Bacillus, Rummeliibacillus, Closlridium, and Paenibacillus. The results showed that all of these strains were able to produce diverse organic compounds, consisting of alcohols, ethers, aldehydes, ketones, acids, aromatic compounds, small amounts pf heterocyclic compounds and their derivatives.To investigate the influences of long-term batch brewing on the production of aromatic compounds, we compared the volatile compositions in the original liquors produced from the 1-year-old and 10-year-old cellars using the GC/MS techniques. There are many volatile compounds in liquors, including various esters, alcohols, fatty acids, aldehydes, acetals, ketones, lactones, furan derivatives and heterocyclic compounds, of which esters, alcohols and fatty acids are the major determinants for the strong aroma style Chinese liquors. Based on their peak area values, low-molecular-weight esters, such as ethyl lactate and ethyl hexanoate were more abundant in the liquors produced in the 10-year-old cellar, whereas some higher-molecular-weight esters, such as ethyl palmitate, ethyl linoleate, and ethyl oleate were more abundant in the liquors from the 1-year-old cellar. The differences among these substances may be one of the factors affecting the quality of Chinese liquors.There are many reports related to the pit-mud microbial ecology. However, limited by disadvantages of employed techniques and complexity of the microbial community in the pit mud, the structure, functional patterns and interactions of the microorganisms in the pit mud still remains unknown, and thus poor improvements have been achieved of the brewing process.In this study, by pyrosequencing the 16S rRNA gene, we found that the microflora in a 10-year-old cellar and a 1-year-old cellar significantly reduced the differences of microbial communities between the top, middle and bottom mud layers, but became surprisingly more diverse in the old cellar than that in the new cellar. Gas chromatography-mass spectrometry assays indicated that the liquors produced in the old cellar contained higher concentrations of certain ester ingredients that are important for the liquor flavor. We further demonstrated that different bacterial isolates from cellar mud were all able to produce diverse organic compounds. These results indicate that long-term spontaneous batch-fermentation accumulates diverse adaptive microbes, which comprehensively produce more flavorful Chinese liquors. |
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