The impact of quorum sensing on the virulence of Aeromonas hydrophila and Aeromonas salmonicida towards burbot (Lota lota L.) larvae

In this study, the link between quorum sensing in Aeromonas spp. and its virulence towards burbot (Lota lota) was investigated. High mortality occurred in burbot juveniles challenged with Aeromonas salmonicida HN-00, but not in juveniles challenged with Aeromonas hydrophila AH-1N. Meanwhile, both A. hydrophila AH-1N and A. salmonicida HN-00 were virulent towards larvae. The effect of quorum sensing on the virulence of A. hydrophila AH-1N towards burbot larvae was further investigated using quorum sensing mutants (N-(butyryl)-L-homoserine lactone production and receptor mutants). Challenge with these mutants resulted in higher survival of burbot larvae when compared to challenge with the wild type, and the addition of the signal molecule N-butyryl-L-homoserine lactone restored the virulence of the quorum sensing production mutant. Moreover, quorum sensing inhibitors protected the burbot larvae from both Aeromonas strains. Finally, the freshwater micro-algae Chlorella saccharophila and Chlamydomonas reinhardtii, which are able to interfere with quorum sensing, also protected burbot from the pathogens. However, QS interference was unlikely to be the only mechanism. This study revealed that the virulence of Aeromonas spp. towards burbot is regulated by quorum sensing and that quorum sensing inhibitors and micro-algae are promising biocontrol agents.     

病原菌致病性胞外产物(ECP)的研究进展

许多病原菌能分泌多种胞外产物,如蛋白酶、磷脂酶、脂肪酶、明胶酶及溶血素等。微生物分泌的明胶酶、蛋白酶、淀粉酶、磷脂酶属于胞外酶,在细菌致病中可以和溶血毒素一起进行协同作用,分解破坏宿主组织成分。笔者综述了病原菌的致病性胞外产物,以及研究胞外产物的方法。     

Construction and analysis of a Mannheimia haemolytica A1 luxS mutant

Mannheimia haemolytica A1 is the causative agent of bovine pneumonic pasteurellosis, a major cause of sickness, death, and economic loss to the feedlot cattle industry. M. haemolytica A1 produces autoinducer-2 (AI-2) like molecules that are capable of inducing quorum sensing system 2 of Vibrio harveyi. This interspecies quorum sensing system has been shown to regulate the expression of virulence genes in several pathogenic bacteria. The protein central to the production of AI-2 is LuxS. To determine if quorum sensing is involved in the regulation of virulence genes in M. haemolytica A1, a luxS mutant was constructed by replacing luxS with a cat cassette. This mutant was verified by PCR analysis, Southern hybridization, as well as its inability to induce bioluminescence in the V. harveyi reporter strain. RT-PCR analysis showed there was no difference in leukotoxin (lktC) mRNA levels, however there were increased mRNA levels of putative virulence associated genes, transferrin binding protein B (tbpB), adhesin (ahs) and capsule biosynthesis (nmaA). Electron microscopy showed that the level of encapsulation in the mutant is higher than the parent. Additionally, the mutant was slightly more adherent to bovine tracheal cells than the parent. In vitro competition assays showed the mutant out-competed the parent under iron-restricted conditions. However, in a calf challenge, the parent was the dominant isolate recovered.     

Flagellin and F4 fimbriae have opposite effects on biofilm formation and quorum sensing in F4ac+ enterotoxigenic Escherichia coli

Bacteria that form biofilms are often highly resistant to antibiotics and are capable of evading the host immune system. To evaluate the role of flagellin and F4 fimbriae on biofilm formation by enterotoxigenic Escherichia coli (ETEC), we deleted the fliC (encoding the major flagellin protein) and/or the faeG (encoding the major subunit of F4 fimbriae) genes from ETEC C83902. Biofilm formation was reduced in the fliC mutant but increased in the faeG mutant, as compared with the wild-type strain. The expression of AI-2 quorum sensing associated genes was regulated in the fliC and faeG mutants, consistent with the biofilm formation of these strains. But, deleting fliC and/or faeG also inhibited AI-2 quorum sensing activity.     

Contribution of AhyR to virulence of Aeromonas hydrophila J-1

Aeromonas hydrophila is a broad-host-range pathogen and its pathogenesis is multifactorial. A regulatory mechanism known as quorum sensing has been found to be involved in the regulation of virulence in many bacteria. In A. hydrophila the ahyR gene encodes LuxR-type response regulator. Here we describe the inactivation of the ahyR gene of A. hydrophila J-1 by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila J-1 by means of the suicide plasmid pJP5603. Cytotoxic effects on EPC cells assay and LD(50) determinations in fish demonstrated that the ahyR mutant was highly attenuated relative to the wild-type strain. Compared with the parent strain, some characteristics, such as biochemical characters and outer membrane protein profiles, had changed. Some main virulent determinants could not be detected, including proteases, amylase, Dnase, hemolysin and S layer. This article confirmed the important function of AhyR in the pathogenesis of A. hydrophila J-1.     

青海玉树不同海拔高度草毡土微生物数量及影响因子

应用稀释平板法探讨了不同海拔高度土壤细菌、真菌和放线菌数量与生境的关系.结果表明,不同海拔样地真菌数量差异显著;AM-2样地细菌、真菌数量最大;AM-3的放线菌数量最大;而AM-4的细菌数量、真菌数量、放线菌数量均为最小值;相关性分析表明,细菌、真菌、放线菌与海拔、地下生物量呈显著正相关;与土壤含水量呈极显著负相关.这表明,土壤微生物受到地下生物量及土壤含水量影响最大.     

Depleting proteins from the growth medium of Mycoplasma capricolum unmasks bacterium-derived enzymatic activities

Mycoplasma constitutes a unique group of bacteria best characterized as lacking peptidoglycan and having one of the smallest genomes of all free-living prokaryotes. Members of this group also represent important pathogens of humans, animals, and plants. Our understanding of the interaction between these pathogens and their hosts is limited, partly due to our inadequate knowledge of the secreted enzymes and virulence factors of these pathogens. Analysis of secreted proteins of mycoplasma has been hampered by their fastidious growth requirements where protein-rich growth supplements are required. Simple ultrafiltration of the complete medium through a 10 kDa cut-off membrane successfully removed virtually all of the polypeptides in the medium and supported the growth of Mycoplasma capricolum (type California kid). This modification (AM medium) exposed the activities of a number of enzymes produced by this bacterium during growth including; acid and alkaline phosphatase, gelatinase, and β-lactamase activities. We also show that the spent culture medium contained hemolysin activity.     

Soft wheat instead of hard wheat in pelleted diets results in high starch digestibility in broiler chickens

(1) The aim of the experiment was to re-examine variations in digestibilities of food components in 3-week-old broiler chickens fed on pelleted diets based on wheats differing in lipase activity and hardness. Fourteen wheat (Triticum aestivum) samples, each from a different cultivar, were included at 550 g/kg in 14 different diets given to male Ross broiler chicks from 7 d of age. The other main ingredients consisted of soyabean meal (353 g/kg) and rapeseed oil (55 g/kg). A 15th diet containing durum wheat (Triticum durum) was also tested. (2) Hardness of wheats (Triticum aestivum) varied between 14 (very soft) and 88 (very hard), and lipase activity of wheats varied from 1 to 13.1 (relative scale). No significant correlation was found between in vitro viscosities and other parameters such as hardness, particle size of wheat flours and lipase. Hardness was correlated with the mean particle size of wheat flours and durability of pellets. (3) Individual lipid digestibilities were negatively correlated with in vitro viscosities of wheats. (4) Individual starch digestibilities were negatively correlated with wheat hardness, particle size of wheat flour before pelleting, and pellet durability. The ratio of measured AME(N) to predicted AME(N) was also negatively correlated with wheat hardness. Simple regression calculation showed that a 100-point increase in wheat hardness resulted in a 3% decrease in the AME(N) value of diets. Multiple regression calculation showed the food/gain ratio (d 10 to d 21) to be positively related to wheat hardness and negatively related to pellet durability. (5) Wheat lipase activity was positively correlated with individual starch digestibility, which was the reverse of a result obtained in a previous experiment. Thus, wheat lipase activity did not seem consistent for predicting starch digestibility and AME(N) values. (6) Among all wheat samples, durum wheat showed the highest protein content and the lowest content of water-insoluble cell-wall. Starch digestibility of durum wheat tended to be lower than that of other wheats (0.916 vs 0.936). However, no significant difference in AME(N) was observed between the durum wheat sample and other wheats. (7) Gut morphometric data measured at d 24 did not show significant differences between dietary treatments.     

Signature-tagged mutagenesis of Vibrio vulnificus

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.     

Hyaluronidase is not essential for the lethality of Erysipelothrix rhusiopathiae infection in mice

To investigate the role of hyaluronidase in the pathogenicity of Erysipelothrix rhusiopathiae, transposon Tn916 was transferred from Enterococcus faecalis CG110 to a virulent strain of E. rhusiopathiae, and hyaluronidase-deficient mutants were isolated. A virulence assay in the mice showed that of the seven hyaluronidase-deficient mutants tested, six mutants were avirulent, but that one mutant, designated AST121, was as virulent as its parental strain. Western immunoblotting with a monoclonal antibody specific to the capsule, a major virulence factor of the organism, revealed that all of the avirulent mutants had lost the capsular antigen, whereas the mutant AST121 did not. These results suggest that the lack of virulence of the six hyaluronidase-negative mutants could be due to a loss of the capsule and that hyaluronidase does not contribute to the lethality of E. rhusiopathiae infection in mice.     

Role of Campylobacter jejuni potential virulence genes in cecal colonization

Campylobacter jejuni, a common commensal in chickens, is one of the leading causes of bacterial gastroenteritis in humans worldwide. The aims of this investigation were twofold. First, we sought to determine whether mutations in the C. jejuni ciaB and pldA virulence-associated genes impaired the organism's ability to colonize chickens. Second, we sought to determine if inoculation of chicks with C. jejuni mutants could confer protection from subsequent challenge with the C. jejuni wild-type strain. The C. jejuni ciaB gene encodes a secreted protein necessary for the maximal invasion of C. jejuni into cultured epithelial cells, and the pldA gene encodes a protein with phospholipase activity. Also included in this study were two additional C. jejuni mutants, one harboring a mutation in cadF and the other in dnaJ, with which we have previously performed colonization studies. In contrast to results with the parental C. jejuni strain, viable organisms were not recovered from any of the chicks inoculated with the C. jejuni mutants. To determine if chicks inoculated with the C. jejuni mutants become resistant to colonization by the C. jejuni parental strain upon subsequent challenge, chicks were inoculated either intraperitoneally (i.p.) or both orally and i.p. with the C. jejuni mutants. Inoculated birds were then orally challenged with the parental strain. Inoculation with the C. jejuni mutants did not provide protection from subsequent challenge with the wild-type strain. In addition, neither the C. jejuni parental nor the mutant strains caused any apparent morbidity or mortality of the chicks. We conclude that mutations in genes cadF, dnaJ, pldA, and ciaB impair the ability of C. jejuni to colonize the cecum, that chicks tolerate massive inoculation with these mutant strains, and that such inoculations do not provide biologically significant protection against colonization by the parental strain.     

Review on the Prohibitive Effect of Gradients in Chinese Traditional Herbs on Hemolysin in Bacteria

Antimicrobial agents play a crucial role in the prevention and treatment of bacterial diseases. However, the widely clinical use of antibiotics makes the bacteria antibiotic-resistance problem much more severe, which could increase the difficulty of clinical treatment. Recently, the method of using traditional Chinese herbs to control bacterial infection by suppressing the bacterial virulence factors has got researchers'attention around the world. Hemolysin is one of the crucial virulence factors in many kinds of bacteria, therefore hemolysin is an ideal target to combat the infection caused by bacteria. The suppressing effect of Chinese herbs to the hemolysin is synthesized and reviewed here to lay theoretical foundation for the clinical treatment of bacterial diseases.     

中药有效成分对细菌溶血素的抑制作用

在防制细菌性疾病方面,抗菌药物起着非常重要的作用,但是随着抗菌药物在临床上的广泛应用,细菌的耐药问题日益突出,给临床治疗带来极大困难。近年来,以中药抑制细菌毒力因子控制细菌感染的治疗策略已引起国内外学者的广泛关注。细菌溶血素是细菌产生的主要毒力因子之一,随着研究的不断深入,开发溶血素抑制剂成为解决细菌感染问题的关键。现针对中药对细菌溶血素的抑制作用进行综述,以期为临床治疗细菌性疾病提供理论依据。     

Studies on Aspergillus Fumigatus; Properties of Intracellular Proteinase

Mycelial filtrates from Aspergillus fumigatus (AF) hydrolyzed protein substrate buffered at various pH values. Using casein as substrate there were distinct activity optima at pH 2.9, pH 6.2, and pH 10, with maximum activity at pH 6.2. Using haemoglobin as substrate there were activity optima at pH 3.6, pH. 4.6, and pH 10, with the biggest activity peak at pH 4.6.The pH stability at 4°G of the caseinase activity at pH 6.2 and pH 10 was strongest at pH 4, common to both, whereas the caseinase activity at pH 2.9 showed maximum pH stability at pH 6—7.The casein hydrolyzing activity at pH 2.9, pH 6.2, and pH 10 showed different optimum incubation temperatures and irregular heat inactivation.Normal rabbit serum inhibited the caseinase activity at pH 2.9 and pH 6.2 to some extent. The caseinase activity at pH 10 was almost completely inhibited. Antiserum against mycelial filtrate showed no definite inhibition beyond that exerted by normal serum.Following electrophoresis of antiserum, the presence of specific neutralizing antibodies against the casein precipitating enzyme of mycelial filtrate from AF could be established. Investigations of 14 AF strains showed immunological uniformity with respect to the casein precipitating enzyme.     

Construction, characterization, and evaluation of the vaccine potential of three genetically defined mutants of avian pathogenic Escherichia coli

The delta galE, delta purA, and delta aroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis. The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively. The delta purA and delta aroA mutants did not grow on minimal medium, whereas the delta galE mutant grew on minimal medium but was sensitive to galactose-induced lysis. The reversion frequencies of all three mutants were <10(-12). The mutants were highly attenuated for virulence as determined by administration of approximately 10(7) colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route. Chickens were vaccinated with the mutants by spray (droplet size approximately 20 microm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy. The mutants were found to be safe. Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E. coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay. In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline-treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain. In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain. The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E. coli 4 days later. Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge. This study indicates that the delta galE, delta purA, and delta aroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.     

Research Progress on Biofilms in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is a gram-negative bacteria which can form biofilms.In this review,we summarized the biology mechanism of biofilms in Pseudomonas aeruginosa,including Pseudomonas aeruginosa adhesion,extracellular polysaccharide Psl and Pel,alginate participated in the biofilm maturation,and quorum sensing systems regulated the formation of biofilm in Pseudomonas aeruginosa,and the drug target on biofilm.The bacterial biofilms protected the bacteria from unsuitable environmental conditions.Through researching the structure and the pathogenesis of Pseudomonas aeruginosa,and its resistance molecular mechanisms,we could adjust or regulate the expression of biofilm-associated factors,optimize the anti-infection treatment in Pseudomonas aeruginosa.     

The germfree murine animal: an important animal model for research on the relationship between gut microbiota and the host

The genomic island 3 (GI-3) shared by Brucella melitensis and Brucella abortus contains 29 genes encoding mostly unknown proteins. Within this island, the open reading frames (ORFs) BAB1_0278 and BAB1_0263 are present, BAB1_0278 encodes a hypothetical protein of 64 amino acids sharing a domain with the GcrA superfamily, whereas the amino acid sequence of BAB1_0263 showed 42% identity with an iron regulated Lsr2 protein. We obtained one deletion mutant for each one of these ORFs present within the B. abortus GI-3 named BA-278 and BA-263, respectively. Both mutants were evaluated with respect to their ability to invade and replicate in nonprofessional and professional phagocytes (HeLa and J774.A1 cells) and their virulence in mice. Both mutants invaded efficiently HeLa and J774. A1 cells, however, 48-h post-infection the BA-278 mutant showed a lower intracellular persistence. The deletion of the ORF BAB1_0278, also affected the persistence of B. abortus in the spleens of mice, unlike to the deletion of the ORF BAB1_0263. These results allow us to conclude that BAB1_0278 ORF contributes to virulence of Brucella, since it is necessary to establish an optimal infectious process.