Bile salts induce resistance to polymyxin in enterohemorrhagic Escherichia coli O157:H7

Many enteric bacteria use bile as an environmental cue to signal resistance and virulence gene expression. Microarray analysis of enterohemorrhagic Escherichia coli O157:H7 (EHEC) treated with bile salts revealed upregulation of genes for an efflux system (acrAB), a two-component signal transduction system (basRS/pmrAB), and lipid A modification (arnBCADTEF and ugd). Bile salt treatment of EHEC produced a basS- and arnT-dependent resistance to polymyxin.     

胆汁分离细菌种类及主要分离菌株耐药性分析

目的了解2000年至2008年临床胆汁普通培养分离细菌的种类,并分析主要分离菌株的耐药性。方法回顾性分析近9年710例胆囊炎和胆囊结石患者胆汁普通培养分离细菌的分布情况及耐药率;用微量稀释法进行药物敏感性测定。结果710例患者胆汁培养共分离出细菌435株,检出率为61.27%,前5位分别为大肠埃希菌、肠球菌、假单胞菌、肺炎克雷伯菌和凝固酶阴性葡萄球菌。革兰阴性杆菌对亚胺培南、阿米卡星的耐药率均低于10%,对常用青霉素类和头孢菌素类抗生素耐药率超过50%。结论胆汁分离细菌以肠道细菌为主,阿米卡星可以作为经验治疗的首选药物。     

MiniReview: bioinformatic study of bile responses in Campylobacterales

Campylobacter, Helicobacter and Wolinella are genera of the order Campylobacterales, belonging to the class Epsilonproteobacteria. Their habitats are various niches in the gastrointestinal tract of higher animals, where they may come into contact with bile. Microorganisms in these environments require mechanisms of resistance to the surface-active amphipathic molecules with potent antimicrobial activities present in bile. This review summarizes current knowledge on the molecular responses to bile by Campylobacterales and other bacterial species that inhabit the intestinal tract and belong to the phyla Proteobacteria, Bacteriodetes, Firmicutes and Actinobacteria. To date, 125 specific genes have been implicated in bile responses, of which 10 are found in Campylobacterales. Genome database searches, analyses of protein sequence and domain similarities, and gene ontology data integration were performed to compare the responses to bile of these bacteria. The results showed that 33 proteins of bacteria belonging to the four phyla had similarities equal to or greater than 50-46% proteins of Campylobacterales. Domain architecture analyses revealed that 151 Campylobacterales proteins had similar domain composition and organization to 60 proteins known to participate in the tolerance to bile in other bacteria. The proteins CmeB, CmeF and CbrR of Campylobacter jejuni involved in bile tolerance were homologous to 42 proteins identified in the Proteobacteria, Bacteriodetes and Firmicutes. On the other hand, the proteins CiaB, CmeA, CmeC, CmeD, CmeE and FlaAsigma(28) also involved in the response to bile of C. jejuni, did not have homologues in other bacteria. Among the bacteria inhabiting the gastrointestinal tract, the Campylobacterales seem to have evolved some mechanisms of bile resistance similar to those of other bacteria, as well as other mechanisms that appear to be characteristic of this order.     

A Product of Heme Catabolism Modulates Bacterial Function and Survival

Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI) tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic E. coli (EHEC), a Gram-negative that causes life-threatening intestinal infections, and E. faecalis, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS) through the absorption of free radicals. In contrast, E. faecalis was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including B. cereus and S. aureus. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome) and Gram-positive bacteria being susceptible to membrane disruption (negative outcome). This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.     

Remodelling of the Vibrio cholerae membrane by incorporation of exogenous fatty acids from host and aquatic environments

The Gram-negative bacteria Vibrio cholerae poses significant public health concerns by causing an acute intestinal infection afflicting millions of people each year. V. cholerae motility, as well as virulence factor expression and outer membrane protein production, has been shown to be affected by bile. The current study examines the effects of bile on V. cholerae phospholipids. Bile exposure caused significant alterations to the phospholipid profile of V. cholerae but not of other enteric pathogens. These changes consisted of a quantitative increase and migratory difference in cardiolipin, decreases in phosphatidylglycerol and phosphatidylethanolamine, and the dramatic appearance of an unknown phospholipid determined to be lyso-phosphatidylethanolamine. Major components of bile were not responsible for the observed changes, but long-chain polyunsaturated fatty acids, which are minor components of bile, were shown to be incorporated into phospholipids of V. cholerae. Although the bile-induced phospholipid profile was independent of the V. cholerae virulence cascade, we identified another relevant environment in which V. cholerae assimilates unique fatty acids into its membrane phospholipids - marine sediment. Our results suggest that Vibrio species possess unique machinery conferring the ability to take up a wider range of exogenous fatty acids than other enteric bacteria.     

胆汁酸代谢与肠道微生物

肠道微生物与胆汁酸代谢密切相关,肠道微生物参与了胆汁酸在肠道中的修饰过程;肠道微生物通过法尼醇受体影响胆汁酸的合成;肠道微生物通过调节胆汁酸的代谢影响机体健康,反过来胆汁酸也可以通过调节肠道微生物菌群的组成影响机体健康.肠道微生物与胆汁酸代谢间的稳态影响着机体健康,现对肠道微生物与胆汁酸代谢及其相互影响做一综述.     

分子伴侣HdeA与HdeB的作用机制

分子伴侣能够与其他蛋白质的不稳定构象相结合并使其稳定．它的功能之一是能够帮助蛋白质进行正确的折叠与组装．最新研究发现,在肠道致病菌的周质空间中存在着酸性条件下能帮助周质蛋白复性的分子伴侣HdeA和HdeB．HdeA在极端酸性的胃部环境中由二聚体迅速解离成具有伴侣活性的单体,HdeA单体能够和变性的底物蛋白结合防止它们酸诱导聚集,从而保护肠道致病菌安全到达肠道．本文对肠道致病菌的耐酸机制进行了总结,最后对 HdeA和HdeB作用机制的研究近况进行综述,最后对HdeA和HdeB以后的研究方向进行了展望．     

Modeling the Infection Dynamics of Bacteriophages in Enteric Escherichia coli: Estimating the Contribution of Transduction to Antimicrobial Gene Spread

Animal-associated bacterial communities are infected by bacteriophages, although the dynamics of these infections are poorly understood. Transduction by bacteriophages may contribute to transfer of antimicrobial resistance genes, but the relative importance of transduction among other gene transfer mechanisms is unknown. We therefore developed a candidate deterministic mathematical model of the infection dynamics of enteric coliphages in commensal Escherichia coli in the large intestine of cattle. We assumed the phages were associated with the intestine and were predominantly temperate. Model simulations demonstrated how, given the bacterial ecology and infection dynamics, most (>90%) commensal enteric E. coli bacteria may become lysogens of enteric coliphages during intestinal transit. Using the model and the most liberal assumptions about transduction efficiency and resistance gene frequency, we approximated the upper numerical limits (“worst-case scenario”) of gene transfer through specialized and generalized transduction in E. coli by enteric coliphages when the transduced genetic segment is picked at random. The estimates were consistent with a relatively small contribution of transduction to lateral gene spread; for example, generalized transduction delivered the chromosomal resistance gene to up to 8 E. coli bacteria/hour within the population of 1.47 × 10⁸ E. coli bacteria/liter luminal contents. In comparison, the plasmidic bla_CMY-2 gene carried by ∼2% of enteric E. coli was transferred by conjugation at a rate at least 1.4 × 10³ times greater than our generalized transduction estimate. The estimated numbers of transductants varied nonlinearly depending on the ecology of bacteria available for phages to infect, that is, on the assumed rates of turnover and replication of enteric E. coli.     

Chaperone-dependent mechanisms for acid resistance in enteric bacteria

The extremely acidic environment of the mammalian stomach not only serves to facilitate food digestion but also acts as a natural barrier against infections of food-borne pathogens. Many pathogenic bacteria, such as enterohemorrhagic Escherichia coli, can breach this host defense and cause severe diseases. These pathogens have evolved multiple intricate strategies to overcome the bactericidal activity of acids. In particular, recent studies have uncovered the central roles of two periplasmic chaperones, HdeA and HdeB, in protecting enteric bacteria from extremely acidic conditions. Here, we review recent advances in the understanding of the acid resistance mechanisms of Gram-negative bacteria and focus on the mechanisms of HdeA and HdeB in preventing acid-induced protein aggregation and facilitating protein refolding following pH neutralization.     

A single‐component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB–TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single‐component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single‐component MdtM transporter and the tripartite AcrAB–TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H⁺ antiport.     

Long-term shifts in patterns of antibiotic resistance in enteric bacteria

Several mechanisms are responsible for the ability of microorganisms to tolerate antibiotics, and the incidence of resistance to these compounds within bacterial species has increased since the commercial use of antibiotics became widespread. To establish the extent of and changes in the diversity of antibiotic resistance patterns in natural populations, we determined the MICs of five antibiotics for collections of enteric bacteria isolated from diverse hosts and geographic locations and during periods before and after commercial application of antibiotics began. All of the pre-antibiotic era strains were susceptible to high levels of these antibiotics, whereas 20% of strains from contemporary populations of Escherichia coli and Salmonella enterica displayed high-level resistance to at least one of the antibiotics. In addition to the increase in the frequency of high-level resistance, background levels, conferred by genes providing nonspecific low-level resistance to multiple antibiotics, were significantly higher among contemporary strains. Changes in the incidence and levels of antibiotic resistance are not confined to particular segments of the bacterial population and reflect responses to the increased exposure of bacteria to antimicrobial compounds over the past several decades.     

Adaptation and response of Bifidobacterium animalis subsp. lactis to bile: a proteomic and physiological approach

Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.     

Effect of bile on Vibrio parahaemolyticus.

Many enteric pathogens are thought to enter a viable but nonculturable state when deprived of nutrients. Virulent strains of the enteric pathogen Vibrio parahaemolyticus are rarely isolated from their low-nutrient aquatic environments, possibly due to their nonculturability. Host factors such as bile may trigger release from dormancy and increase virulence in these strains. In this study, the addition of bile or the bile acid deoxycholic acid to estuarine water-cultured bacteria led to an increase in the direct viable count and colony counts among the virulent strains. This effect was not demonstrated in the nonvirulent strains, and it was reversed by extraction of bile acids with cholestyramine. Bile-treated V. parahaemolyticus had lower levels of intracellular calcium than untreated cells, and this effect coincided with an increase in the number of metabolically active cells. Chelation of intracellular calcium with BAPTA/AM (R. Y. Tsien, Biochemistry 19:2396-2402, 1980) produced similar results. Addition of bile to V. parahaemolyticus cultures in laboratory medium enhanced factors associated with virulence such as Congo red binding, bacterial capsule size, and adherence to epithelial cells. These results suggest that a bile acid-containing environment such as that found in the human host favors growth of virulent strains of V. parahaemolyticus and that bile acids enhance the expression of virulence factors. These effects seem to be mediated by a decrease in intracellular calcium.     

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

We examined three groups of wild baboons (Papio cynocephalus) in Amboseli National Park, Kenya, to determine the prevalence of aerobic antibiotic-resistant fecal bacteria in nonhuman primates with and without contact with human refuse. Using standard isolation and replica plating techniques, we found only low numbers of antibiotic-resistant gram-negative enteric bacteria in two groups of baboons leading an undisturbed existence in their natural habitat and having limited or no contact with humans. However, resistance was significantly higher among enteric bacteria from the third group of baboons living in close proximity to a tourist lodge and having daily contact with unprocessed human refuse. Conjugation studies and analysis of the cell DNA by gel electrophoresis showed that in many cases resistance was plasmid-borne and transferable. These data suggest that wild nonhuman primates in frequent contact with human debris have a higher proportion of antibiotic-resistant enteric bacteria than do conspecifics without this contact. The findings further suggest that such groups of wild animals may constitute a heretofore overlooked source of antibiotic resistance in the natural environment.     

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

We examined three groups of wild baboons (Papio cynocephalus) in Amboseli National Park, Kenya, to determine the prevalence of aerobic antibiotic-resistant fecal bacteria in nonhuman primates with and without contact with human refuse. Using standard isolation and replica plating techniques, we found only low numbers of antibiotic-resistant gram-negative enteric bacteria in two groups of baboons leading an undisturbed existence in their natural habitat and having limited or no contact with humans. However, resistance was significantly higher among enteric bacteria from the third group of baboons living in close proximity to a tourist lodge and having daily contact with unprocessed human refuse. Conjugation studies and analysis of the cell DNA by gel electrophoresis showed that in many cases resistance was plasmid-borne and transferable. These data suggest that wild nonhuman primates in frequent contact with human debris have a higher proportion of antibiotic-resistant enteric bacteria than do conspecifics without this contact. The findings further suggest that such groups of wild animals may constitute a heretofore overlooked source of antibiotic resistance in the natural environment.     

Long-Term Shifts in Patterns of Antibiotic Resistance in Enteric Bacteria

Several mechanisms are responsible for the ability of microorganisms to tolerate antibiotics, and the incidence of resistance to these compounds within bacterial species has increased since the commercial use of antibiotics became widespread. To establish the extent of and changes in the diversity of antibiotic resistance patterns in natural populations, we determined the MICs of five antibiotics for collections of enteric bacteria isolated from diverse hosts and geographic locations and during periods before and after commercial application of antibiotics began. All of the pre-antibiotic era strains were susceptible to high levels of these antibiotics, whereas 20% of strains from contemporary populations of Escherichia coli and Salmonella enterica displayed high-level resistance to at least one of the antibiotics. In addition to the increase in the frequency of high-level resistance, background levels, conferred by genes providing nonspecific low-level resistance to multiple antibiotics, were significantly higher among contemporary strains. Changes in the incidence and levels of antibiotic resistance are not confined to particular segments of the bacterial population and reflect responses to the increased exposure of bacteria to antimicrobial compounds over the past several decades.     

The interaction between bacteria and bile

Commensal and pathogenic microorganisms must resist the deleterious actions of bile in order to survive in the human gastrointestinal tract. Herein we review the current knowledge on the mechanisms by which Gram-positive and Gram-negative bacteria contend with bile stress. We describe the antimicrobial actions of bile, assess the variations in bile tolerance between bacterial genera and examine the interplay between bile stress and other stresses. The molecular mechanisms underlying bile tolerance are investigated and the relationship between bile and virulence is examined. Finally, the potential benefits of bile research are briefly discussed.     

胆总管结石患者胆汁病原菌分布特点及耐药性分析

目的:探讨胆总管结石患者胆汁病原菌的分布特点以及耐药性的分析。方法:选择2016年6月-2017年6月期间我院收治的胆总管结石合并胆道感染患者160例为研究对象,所有患者均进行逆行内镜胰胆管造影(ERCP)并抽取胆汁标本,进行细菌培养和耐药性实验,评价分析胆汁病原菌的分布特点及耐药性情况。结果:160例患者中有117例(73.13%)检出病原菌,共培养出病原菌130株,其中有13例患者为两种病原菌同时感染。革兰阴性菌有95株(73.08%)、革兰阳性菌有31株(23.85%)、真菌有4株(3.08%)。比例由高到低的前六位病原菌依次为:大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌、屎肠球菌、铜绿假单胞菌、粪肠球菌。革兰阴性菌对亚胺培南、阿米卡星、美罗培南、他唑巴坦、头孢吡肟等三四代头孢菌素耐药率较低,对头孢曲松、环丙沙星、左氧氟沙星、哌拉西林、氨苄西林等耐药率较高。革兰阳性菌对替拉考宁、万古霉素、利奈唑胺等耐药率较低,对四环素、环丙沙星、左氧氟沙星、克林霉素、氨苄西林等耐药率较高。真菌对酮康唑、伊曲康唑、氟康唑等耐药率较低,对两性霉素B耐药率较高。结论:胆总管结石患者胆汁病原菌主要为革兰阴性菌、其次为革兰阳性菌,各病原菌对各种抗菌药物表现出不同的耐药性,因此在临床治疗时应参考药敏试验结果进行合理选择治疗药物。     

Conserved amphiphilic feature is essential for periplasmic chaperone HdeA to support acid resistance in enteric bacteria

The extremely acidic environment of the mammalian stomach (pH 1-3) represents a stressful challenge for enteric pathogenic bacteria, including Escherichia coli, Shigella and Brucella. The hdeA (hns-dependent expression A) gene was found to be crucial for the survival of these enteric bacteria under extremely low pH conditions. We recently demonstrated that HdeA is able to exhibit chaperone-like activity exclusively within the stomach pH range by transforming from a well-folded conformation at higher pH values (above pH 3) into an unfolded conformation at extremely low pH values (below pH 3). This study was performed to characterize the action mechanisms and underlying specific structural features for HdeA to function in this unfolded conformation. In the present study, we demonstrate that the conserved 'amphiphilic' feature of HdeA, i.e. the exposure of the conserved hydrophobic region and highly charged terminal regions, is essential for exhibiting chaperone-like activity under extremely low pH conditions. Mutations that disrupt this amphiphilic feature markedly reduced the chaperone-like activity of HdeA. The results also strongly suggest that this acid-induced chaperone-like activity of HdeA is crucial for acid resistance of the enteric bacteria. Moreover, our new understanding of this amphiphilic structural feature of HdeA helps to better interpret how this unfolded (disordered) conformation could be functionally active.     

Importance of microbial defence systems to bile salts and mechanisms of serum cholesterol reduction

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.