Study on Differences in Procyanidine Content Between Muscat Hamburg Grape From Different Producing Areas

Wen XU Qiusheng CHEN Zhe YANG Qiang ZHANG Rui SUN Fang SU Yi PEI

AbstractA biofilm refers to a group of organized bacteria attached to the surface of living or inanimate objects and surrounded by some macromolecules. It is rich in organic components such as polysaccharides, peptides and phospholipids, greatly increases the resistance of bacteria to antibacterial drugs, and can cause infections in humans and animals. At present, the measure to prevent the infections is to prevent microbial colonies from adhering to the surface of the objects, which will help to improve the therapeutic effect of clinically serious infectious diseases. In addition, the identification and inhibition of biofilm formation genes is also an important research direction to control such infections. In this paper, molecular mechanisms of bacterial biofilm resistance to drugs that has caused widespread concern were summarized.

Key wordsBiofilm; Quorum sensing; Efflux pump; Drug resistance; Stress response; Molecular mechanism

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Received: October 14, 2018Accepted: November 28, 2018

Supported by Natural Science Foundation of Tianjin City (07JCYBJC16000); Project for Key Technology Integration and Enhancement of Students’ Comprehensive Ability of Affiliated Animal Hospital of Tianjin Agricultural University (ZH004901).

Yanfei LIU (1970-), female, P. R. China, associate professor, master, devoted to research about preventive veterinary medicine.

*Corresponding author. Email: jiandeyang@126.com.

A biofilm (Bf) is composed of a group of microorganisms enveloped in the extracellular polymeric matrix produced by themselves, and it is widely distributed on the surfaces of natural water systems or drinking water systems, living tissues, medical devices, etc［1］. Biofilm matrix is composed of complex proteins, polysaccharides and DNA, and has attracted much attention in respect of resisting antibiotics, phagocytosis and killing roles of other disinfectant components. A biofilm community has unique macroscopic and microscopic structural features in the natural environment. Through biofilms, nutrients, gases and antibacterial substances are diffused, and biofilms can also adjust their structure to accommodate external and internal changes. Because the cells are adjacent, they can exchange different density of sensitive molecules and additional chromosomal plasmids, making each biofilm community unique.

In different environments, microorganisms acquire temporary or permanent biological resistance and may destroy or inhibit other members of the same strain［1］. Antimicrobial resistance is well known, but there is relatively little understanding of resistance to food preservatives, disinfectants and antimicrobial agents. The biofilm resistance of microorganisms is affected by many factors［2］. Bacterial adhesion in food and dairy industries is also a problem caused by the wellknown biofilm mechanism. Antibacterial drugs act on a series of functional genetic material, enzymes, respiratory systems and other cellular sites. However, due to gene exchange and inherent differences, such as bacterial unique capsule components and nonsensitive proteins, different bacteria respond differently to a bacteriacide. Bacterial biofilms enhance drug resistance, which is associated with many persistent infectious diseases. In this paper, the molecular mechanisms of biofilm resistance to drug will be discussed.

External Structure of Bacteria

Capsule

The capsule is an indispensable component of a biofilm, and its thickness is about 0.2-1.0 μm in Grampositive and Gramnegative bacteria. Electrostatics, van der Waals forces and hydrogen bonding cause biofilms to stick to each other or adhere to solid surfaces. The composition of the capsule is flexible and regulated by biofilm growth, which facilitates the survival of pathogenic bacteria in extremely host environments. The resistance of bacteria to antibiotics and antibacterial agents is influenced by the capsule matrix. When the weight of a bacteriacide accounts for 25% of the weight of the capsule, the adsorption site of the capsule matrix limits the transport of the bacteriacide, providing a binding site for adhesion of an external enzyme. The external enzyme has a specific activity of binding the bacteriacide and provide a substrate for the metabolic degradation of the bacteriacide, thereby reducing the efficacy［3］.

Outer membrane structure of a bacterium

Antibacterial drugs must penetrate into the target site to function. The change or adaptation of bacterial capsule is the main reason for bacteria to resist antibiotics. The lipopolysaccharide layer and the bottom phospholipid mainly prevent hydrophilic antibacterial drugs from entering the outer membrane, while the outer membrane protein resists hydrophobic antibiotics. In addition, in the complex cell walls of Grampositive bacteria, a variety of lipids and poreforming proteins combine to form a hydrophilic channel to resist antibiotics. The outer membrane structure of mycobacteria is indispensable in the process of fighting against antibiotics, and some strains resistant to antibiotics lack or overexpress outer membrane proteins. For example, Pseudomonas aeruginosa strains lack Opr D (Opr D is a channel in which isothiazolone and other carbon source compounds selectively enter)［4］.

Growth and Metabolism of Bacteria in Biofilms

The growth rate and metabolic activity of bacteria are affected by differences in nutrients and oxygen in biofilms. The effects of different metabolic matrices and metabolite concentrations on bacterial growth and activity level in biofilms have been confirmed［5］. Under different culture conditions, Clostridium difficile has valuable growth and fermentation information, which leads to heterogeneity of bacterial population. Nutrition and oxygen in the surrounding area of a biofilm accelerate the metabolic activities of the cells, which in turn promotes the proliferation of bacteria. Conversely, due to malnutrition, metabolic potential is limited, resulting in slow growth of cells in the biofilm matrix, which can be attributed to the decreases in the levels of guanosine 3,5′bisphosphate(ppGpp) accumulation and RNA (tRNA and rRNA) synthesis. The heterogeneity of cell metabolism and growth rate is mainly determined by the synthesis of cellular enzymes in biofilms, and the enzymes are affected by changes in the growth cycle of bacteria. In the stationary phase or in the slow growth phase, the synthesis of cellular enzymes of bacteria is inhibited. Biocides kill metabolically active bacteria, while bacteria in the dormant growth stage are less affected by bactericidal drugs. During the dormant growth phase of Escherichia coli, ppGpp inhibits the activity of autolytic enzymes and limits the synthesis of cells［6］. Oxygen in biofilms can affect metabolic activity. In P. aeruginosa, when ciprofloxacin and tobramycin are given, bacteria are killed in pure oxygen, but the decrease in oxygen content enhances the resistance to antibiotics［7］. Bacterial biofilms express specific genes under anaerobic conditions, increasing the level of drug resistance.

The resistance of biofilms to drugs is related to the specific growth of bacteria within them［8］. The effects of a bacteriacide on bacteria depend on the physiological state and nature of the habitat of bacteria. Limited nutrient composition can affect the synthesis of barrier components, which may affect the integrity of the capsule components of thalli. When a biofilm is exposed to the inhibitory concentration of a bactericide, the resistant thalli can exhibit phenotypic adaptation. Heat and starvation stress increase the resistance of E. coli to ultraviolet rays or H2O2. Enterococcus upregulates the expression of antioxidant enzyme genes and downregulates the role of oxidase after the induction of oxidative stress. However, once a bacteriacide is removed, the phenotype of drug resistance will disappear. Some people think that due to the restriction of nutrients in biofilms, bacteria grow slowly and are in the starvation phase. When nutrients are rich and bacteria multiply, nongrowth cells are not susceptible to various antibiotics［9］.

Genetic Factors

Genetic adaptation

Biofilm thalli require genetic adaptation to reduce susceptibility to drugs and to obtain a relatively selfprotecting phenotype. Multidrug operons control the expression of various genes in E. coli and are associated with multidrug resistant phenotypes including antibiotics, organic solvents and other disinfectants. In addition, Mycobacterium tuberculosis can be dormant for decades in a stressful environment, and antituberculosis drugs have no effect on it. Most bacteria are fermentative, produce oxidative degradation and repair enzymes, and exhibit oxidative stress response. When the thalli exhibiting stress response are exposed to subinhibitory factors for several hours, they are more resistant to harmful factors. Recently, multiple defense genes of E. coli have been identified, such as a coding catalyst, superoxide dismutase, hydrogen peroxide reductase, alkyl glutathione reductase, and DNA repair enzyme. In addition, different regulatory genes, such as asoxyR and soxR, determine the intracellular redox potential and activate stress response when cells are oxidized［10］.

Efflux pumps

In an antibacterial environment and other extreme conditions, there is an efflux pump system that promotes bacterial survival. Efflux pumps use the acquired resistance mechanism to resist antibacterial agents, and the excessive production of efflux pumps can lead to multidrug resistance. Grampositive bacteria (bacillus, lactic acid, Staphylococcus aureus, etc.) can also cause serious infection due to drug resistance［11］. They have multiple mechanisms of drug resistance and are capable of transporting a variety of unrelated compounds, resulting in multiple phenotypes of drug resistance. In some pathogenic bacteria, such as E. coli and Klebsiella pneumoniae, efflux pumps reduce the penetration of hydrophobic solutes by decreasing the production of membrane pore proteins, thereby reducing the transmembrane diffusion of lipid solutes. When biofilms are exposed to lower concentrations of antibiotics, such as chloramphenicol, tetracycline, and atypical organisms (such as salicylates and chlorinated phenols), the expression of multiple drug resistant operons and efflux pumps can be induced. Similarly, the multiple resistant phenotypes of E. coli biofilms is regulated by mar and acrAB encoding genes. Some antibiotics, such as penicillin, cephalosporin, rifampicin, nalidixic acid, fluoroquinolone and oxidative stress agents, promote the expression of mar in bacteria, thereby increasing drug resistance［12］. In addition, the sublethal doses of several commonly used drugs, such as tetracycline, chloramphenicol, salicylate and paracetamol, can induce an increase in mar expression［13］.

External Factors

Quorum sensing

Quorum sensing (QS) regulates the interaction between bacterial cells. Studies have found that through the antagonism of receptors, some families of natural compounds can inhibit quorum sensing. Bacteria can sense and respond to increased density of cell population by inducing the expression of specific genes. Quorum sensing involves the production and secretion of an acylhomoserine lactone (AHL) that diffuses from cells into a culture medium through cell walls. In Grampositive bacteria, quorum sensing secretes polypeptides as signaling molecules and utilizes regulatory systems to detect changes in gene expression patterns and polypeptides. In addition, autoinducer2 is another form of quorum sensing mechanism, which is present in Grampositive and negative bacteria［14］. In the formation of a biofilm, the effect of quorum sensing mediated by signalling molecules has been confirmed, and quorum sensing affects the structure of the biofilm and then regulates the synthesis of degrading enzymes. In addition, in a proper nutrient environment, the expression of the phenotype mediated by quorum sensing plays a crucial role in cell migration and also protects neonatal thalli from harmful environments. In Aeromonas, three quorum sensing systems regulate the formation, movement and toxic activity of biofilms［15］. The signal transduction mechanism of P. aeruginosa controls the expression of superoxide dismutase and catalase genes, and the two enzymes mediate the resistance of hydrogen peroxide. The deficiency of quorum sensing is associated with thinner biofilms and lower yield of extracellular polymeric substances (EPS), and this mutation or defect makes biofilms susceptible to kanamycin. Studies indicate that quorum sensing reacts directly or indirectly to environmental stress, which in turn affects the formation of biofilms［16］.

Stress response

The stress response of biofilms is characterized by a large number of changes in bacterial physiology and morphology, which greatly increases the stress resistance of the cells. In general, the stress response of intestinal flora such as E. coli and Salmonella controls the formation of bacterial capsules and the synthesis of fine accumulated pili. In addition, the stress response function is to prevent cell damage rather than repair. Several factors can lead to induced stress, such as nutrient deficiency of bacteria in the stable period, high or low temperature, high permeability and acidic environment. When E. coli is exposed to adverse conditions, it can induce the production of RNA polymerase σ subunit, which controls more than 50 genes that determine stress tolerance. The position of general stress response gene expression sites is relatively stable in biofilm matrix, so that drug resistance increases［17］.

Sustained Infection of bacteria

Persisters belong to drugresistant flora and are often the chief culprit of serious chronic infectious diseases. Detection of persisters is a major clinical challenge. A study on the formation of ATPdependent persisters have shown that low levels of ATP can reduce the targeting activity of antibiotics, leading to the formation of persisters. Biofilms contain persisters resistant to multiple drugs［18］. Grampositive or negative bacteria in the late growth period can produce retaining persisters that are resistant to multiple drugs or antibiotics.

The growth stage of bacterial community controls the formation of persisters, and some of the bacteria rapidly multiply and survive under the action of a lethal dose of antibacterial drugs. At the stationary stage, bacteria produce higher levels of persisters, which is positively correlated with the drug resistance in biofilms［19］. The extracellular polysaccharideprotein matrix protects the persisters in biofilms against immune system. After the antibiotic action disappears in bacterial population, persisters begin to induce the growth of biofilms again［20］. Studies have shown that if bacteria are diluted in the stationary phase, the number of persisters will decrease. The formation of persisters is also dependent on the metabolic activity of bacteria, which may suggest that persisters are a dormant variant of the wild type rather than a mutant. However, persisters do not respond to bacteriacides［18］. Persisters produce multidrug resistant proteins to compete with antibiotics. It is worth noting that antibiotics are bacteriacides that destroys the function of target cells, rather than inhibiting their effects, thereby leading to cell damage. The tolerance of persisters is related to apoptosis, and the action of antibacterial compounds leads to cell damage rather than complete cell death, which indirectly triggers apoptosis. Bacterial autolysis is also seen in biofilms, which is caused by peptidoglycan hydrolase［21］.

Agricultural Biotechnology2019

Conclusions and Outlooks

From the aspects of external structure, bacterial growth and metabolism, genetic factors, external factors and persister formation, the mechanism of biofilm resistance to drugs was expounded to provide reference for the future development and treatment of biofilm bacterial infection. Some signaling molecule analogs (such as AHLs analogs) or natural products and their derivatives (such as imidazoles and anthraquinone derivatives) may be used to potentially treat biofilm bacterial diseases. New antibiofim preparations should have the characteristics of high efficiency and low toxicity, and reduce the selection pressure of microorganisms, thereby reducing the production of microbial resistance to drugs and prolonging the service life of the drugs［23-24］. With the deepening of research, people will have a new breakthrough in understanding of biofilm resistance to drugs, and hope to achieve complete treatment of biofilm disease at an early date.

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