A medium for the selective isolation of Edwardsiella ictaluri

A selective medium, called Edwardsiella ictaluri medium (EIM), has been formulated for the isolation of Edwardsiella ictaluri. The medium inhibits the growth of most gram-negative bacteria, except Proteus sp., Serratia marcescens and some isolates of Aeromonas hydrophila and Yersinia ruckeri. The bacteria that grow on the EIM are easily differentiated from E. ictaluri based on colony morphology. The EIM inhibits gram-positive bacteria with the exception of enterococci. The addition of fungizone to EIM suppressed the growth of most fungi. The EIM allows the evaluation of environmental reservoirs, levels of contamination and carrier states of E. ictaluri.     

Ichthyophthirius multifiliis as a potential vector of Edwardsiella ictaluri in channel catfish

There is limited information on whether parasites act as vectors to transmit bacteria in fish. In this trial, we used Ichthyophthirius multifiliis and fluorescent Edwardsiella ictaluri as a model to study the interaction between parasite, bacterium, and fish. The percentage (23-39%) of theronts fluorescing after exposure to E.?ictaluri was significantly higher than control theronts (~?6%) using flow cytometry. Theronts exposed to E.?ictaluri at 4?×?10(7) CFU?mL(-1) showed a higher percentage (~?60%) of fluorescent theronts compared to those (42%) exposed to 4?×?10(3) CFU?mL(-1) at 4?h. All tomonts (100%) carried the bacterium after exposure to E.?ictaluri. Edwardsiella ictaluri survived and replicated during tomont division. Confocal microscopy demonstrated that E.?ictaluri was associated with the tomont surface. Among theronts released from tomonts exposed to E.?ictaluri, 31-66% were observed with attached E.?ictaluri. Sixty percent of fish exposed to theronts treated with 5?×?10(7) E.?ictaluri?mL(-1) were positive for E.?ictaluri at 4?h as determined by qPCR or fluorescent microscopy. Fluorescent E.?ictaluri were observed on trophonts in skin and gill wet mounts of dead fish. This study demonstrated that Ich could vector E.?ictaluri to channel catfish.     

In vitro inhibitory effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri

AIM: This study was conducted to evaluate the toxic effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri. METHODS AND RESULTS: Inhibitory effect of various concentrations of gossypol on the growth of E. ictaluri was determined. Bacterial recovery was performed by preincubation of bacteria in medium containing various concentrations of gossypol and subsequent activation of bacteria by inoculating on gossypol-free plates. Concentrations of racemic gossypol, (+)-gossypol and (-)-gossypol of 1.5 microg ml(-1) or higher significantly reduced the number of bacterial colonies compared with that of the control. The growth of E. ictaluri was completely inhibited on agar plates supplemented with 3 microg ml(-1), regardless of the forms of gossypol. The inhibitory effect of (+)-gossypol was higher than that of (-)-gossypol or gossypol-acetic acid. Recovery of E. ictaluri was <50% for all three forms of gossypol at concentrations of 5 microg ml(-1). Bacterial recovery remained relatively constant (6.5%) at gossypol concentrations from 10 to 100 microg ml(-1). Complete killing of E. ictaluri was not reached at gossypol levels up to 100 microg ml(-1). CONCLUSION: Gossypol-acetic acid, and (+)- and (-)-optical isomers have anti-bacterial effect against E. ictaluri. The results suggest the action is bacteriostatic rather than bactericidal. SIGNIFICANCE AND IMPACT OF THE STUDY: The therapeutic effect of gossypol against E. ictaluri may be useful in controlling enteric septicaemia of catfish.     

Genome sequence of Edwardsiella ictaluri 93-146, a strain associated with a natural channel catfish outbreak of enteric septicemia of catfish

Edwardsiella ictaluri is the cause of extensive mortalities and economic losses to the channel catfish industry of the southeast United States. Here we report the complete genome of Edwardsiella ictaluri 93-146. Whole-genome sequence analysis of E. ictaluri provides a tool for understanding the genomic regions specific to the species and the Edwardsiella genus.     

■爱德华氏菌变异株C9605及对鳖的致病性研究

从患白板综合症的病鳖分离到一株细菌（C9605）,该菌为革兰氏阴性,直杆状,周生鞭毛。接触酶阳性,氧化酶阴性,还原硝酸盐,对多粘菌素不敏感,不利用柠檬酸盐和丙二酸盐作唯一碳源,不从甘露醇、蔗糖、海藻糖、L-阿拉伯糖产酸。根据这些特性,菌株可归于爱德华氏菌。但是该菌发酵木糖产酸,产生H2S,耐青霉素,故鉴定为爱德华氏菌变异株（Edwardsiellaictalurivariationstrain）。人工感染实验证实,该菌株是鳖白板综合症的病原菌。     

Purification of antimicrobial factor from granules of channel catfish peripheral blood leucocytes

The channel catfish (Ictalurus punctatus) is extensively used in aquaculture in the Southeast US and is susceptible to many bacterial infections acquired from its pond environment. Research is needed to better understand the defensive response and innate immunity of channel catfish against fish pathogens like Edwardsiella ictaluri and Aeromonas hydrophila. The main objectives were purification and characterization of an innate antimicrobial factor isolated from catfish leucocytes that has both bactericidal and antiviral activities. Oxygen-independent mechanisms of innate immunity for killing microorganisms have not been identified in leucocytes of channel catfish. Leucocytes were separated from catfish blood, and granule extracts were obtained by homogenization, centrifugation, and extraction with 10% acetic acid. The granule extracts were further purified by gel filtration chromatography. Bactericidal assays against the two fish pathogens and SDS-PAGE analysis were done on the isolated antimicrobial factor. Determination of antiviral activity of the factor was done by in vitro tissue culture using herpes simplex virus-type 1. Mass spectrometry analyses were done for molecular weight (655 Da), purity, and structural characterization of the innate non-peptide antimicrobial factor.     

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。     

呼吸机相关性肺炎的常见病原菌耐药性分析

目的:探讨呼吸机相关性肺炎(VAP)的常见病原菌并分析其耐药性,为临床治疗提供依据。方法:选取我院收治的135例VAP患者的临床资料,分析其病原菌分布以及抗菌药物的耐药性。结果:135例患者中共分离出183株病原菌,其中革兰氏阴性菌135株(占73.77%),革兰氏阳性细菌33株(占18.03%),真菌15株(占8.20%)。革兰氏阴性菌主要为鲍曼不动杆菌,占35.52%,革兰氏阳性细菌主要为金黄色葡萄球菌,占9.84%,革兰阳性菌无一对万古霉素耐药,除了米诺环素总耐药率为42.42%外,其余病原菌对于常用的药物总耐药率均大于60.0%,革兰阴性菌普遍存在多药耐药现象。结论:引起VAP患者感染的主要致病菌为革兰阴性菌群,且存在严重的多重耐药现象,在临床上应加强对VAP疾病的预防和控制,合理应用抗菌药物。     

Identification of antimicrobial activity among new sulfonamide metal complexes for combating rapidly growing mycobacteria

Mycobacteriosis is a type of infection caused by rapidly growing mycobacteria (RGM), which can vary from localized illness, such as skin disease, to disseminated disease. Amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem and sulfamethoxazole are antimicrobial drugs chosen to treat such illnesses; however, not all patients obtain the cure. The reason why the treatment does not work for those patients is related to the fact that some clinical strains present resistance to the existing antimicrobial drugs; thereby, the research of new therapeutic approaches is extremely relevant. The coordination of antimicrobial drugs to metals is a promising alternative in the development of effective compounds against resistant microorganisms. Sulfonamides complexed with Au, Cd, Ag, Cu, and Hg have shown excellent activity against a variety of microorganisms. Considering the importance of fighting against infections associated with RGM, the objective of this study is to evaluate the antimycobacterial activity of metal complexes of sulfonamides against RGM. Complexed sulfonamides activity were individually tested and in association with trimethoprim. The minimum inhibitory concentration (MIC) and time-kill curve of compounds against the standard strains of RGM [Mycobacterium abscessus (ATCC 19977), Mycobacterium fortuitum (ATCC 6841) and Mycobacterium massiliense (ATCC 48898)] was determined. The interaction of sulfonamides with trimethoprim was defined by inhibitory concentration index fractional for each association. The results showed that sulfonamides complexed whit metals have outstanding antimicrobial activity when compared to free sulfamethoxazole, bactericidal activity and synergistic effect when combined with trimethoprim.     

Synergistic Antibacterial and Anti-Inflammatory Activity of Temporin A and Modified Temporin B In Vivo

Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10–14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and anti-inflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3–6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.     

Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring

Edwardsiella ictaluri is a facultative intracellular bacterium that causes enteric septicemia of catfish (ESC). In this study, we aimed to develop bioluminescent E. ictaluri that can be monitored by noninvasive bioluminescence imaging (BLI). To accomplish this, the luxCDABE operon of Photorhabdus luminescens was cloned downstream of the lacZ promoter in the broad host range plasmid pBBR1MCS4. Edwardsiella ictaluri strain 93-146 transformed with the new plasmid, pAKlux1, was highly bioluminescent. pAKlux1 was stably maintained in E. ictaluri without any apparent effect on growth or native plasmid stability. To assess the usefulness of the bioluminescent strain in disease studies, catfish were infected with 93-146 pAKlux1 by intraperitoneal injection and by bath immersion, and in vivo bacterial dissemination was observed using BLI. This study demonstrated that bioluminescent E. ictaluri can be used for real-time monitoring of ESC in live fish, which should enable observation of pathogen attachment sites and tissue predilections.     

Synthesis, characterization and antimicrobial activity of new aliphatic sulfonamide

A series of novel aliphatic sulfonamide derivatives (1-7) were synthesized and characterized by elemental analyses, FT-IR, (1)H NMR, (13)C NMR and LC-MS techniques. All the synthesized compounds were evaluated in vitro as antimicrobial agents against representative strains of Gram-positive (Staphylococcus aureus ATCC 25953, Bacillus cereus ATCC 6633 and Listeria monocytogenes ATCC Li6 (isolate), Gram-negative bacteria (Escherichia coli ATCC 11230) and antifungal agent against Candida albicans (clinical isolate) by both disc diffusion and minimal inhibition concentration (MIC) methods. All these bacteria and fungus studied were screened against some antibiotics to compare with our chemicals' zone diameters. Our aliphatic sulfonamides have highest powerful antibacterial activity for Gram-negative bacteria than Gram-positive bacteria and antibacterial activity decreases as the length of the carbon chain increases.     

临床病原菌种类及耐药性监测

目的 探讨病原菌种类及其对抗菌药物的耐的耐药状况。方法 收集１９９８年１月－１９９９年１２月临床感染标本分离的病原菌并分析其种类,药敏试验采用Ｋｉｒｂｙ－Ｂａｕｅｒ纸片扩散法。结果 １１８２株病原菌,革兰氏阳性球菌６０４株（５１．１％）,革兰氏阴性杆菌５７８株（４８．９％）。病原菌以金黄一萄球菌、大肠埃希菌、表皮葡萄球菌、铜绿假单胞菌最多见。去甲万古霉素、阿米卡星、新霉素对革兰氏阳性球菌抗菌作用较     

Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Edwardsiella ictaluri

Comparison of the 23S rRNA gene sequences of Edwardsiella tarda and Edw. ictaluri confirmed a close phylogenetic relationship between these two fish pathogen species and a distant relation with the 'core' members of the Enterobacteriaceae family. Analysis of the rrl gene for 23S rRNA in Edw. ictaluri revealed the presence of an intervening sequence (IVS) in helix-45. This new 98bp IVS shared 97% nucleotide identity with Salmonella typhimurium helix-45 IVS. Edw. ictaluri helix-45 IVS was present in all Edw. ictaluri strains analyzed and in at least six rrl operons within each cell. Fragmentation of 23S rRNA due to IVS excision by RNase III was observed by methylene blue staining of ribosomal RNA extracted from Edw. ictaluri isolates. This is the first report of an IVS in the 23S rRNA gene of the genus Edwardsiella.     

Edwardsiella comparative phylogenomics reveal the new intra/inter-species taxonomic relationships, virulence evolution and niche adaptation mechanisms

Edwardsiella bacteria are leading fish pathogens causing huge losses to aquaculture industries worldwide. E. tarda is a broad-host range pathogen that infects more than 20 species of fish and other animals including humans while E. ictaluri is host-adapted to channel catfish causing enteric septicemia of catfish (ESC). Thus, these two species consist of a useful comparative system for studying the intricacies of pathogen evolution. Here we present for the first time the phylogenomic comparisons of 8 genomes of E. tarda and E. ictaluri isolates. Genome-based phylogenetic analysis revealed that E. tarda could be separate into two kinds of genotypes (genotype I, EdwGI and genotype II, EdwGII) based on the sequence similarity. E. tarda strains of EdwGI were clustered together with the E. ictaluri lineage and showed low sequence conservation to E. tarda strains of EdwGII. Multilocus sequence analysis (MLSA) of 48 distinct Edwardsiella strains also supports the new taxonomic relationship of the lineages. We identified the type III and VI secretion systems (T3SS and T6SS) as well as iron scavenging related genes that fulfilled the criteria of a key evolutionary factor likely facilitating the virulence evolution and adaptation to a broad range of hosts in EdwGI E. tarda. The surface structure-related genes may underlie the adaptive evolution of E. ictaluri in the host specification processes. Virulence and competition assays of the null mutants of the representative genes experimentally confirmed their contributive roles in the evolution/niche adaptive processes. We also reconstructed the hypothetical evolutionary pathway to highlight the virulence evolution and niche adaptation mechanisms of Edwardsiella. This study may facilitate the development of diagnostics, vaccines, and therapeutics for this under-studied pathogen.     

2011～2015年医院呼吸内科常见革兰阴性菌耐药性分析与临床用药探讨

目的：分析2011～2015年我院呼吸内科常见革兰阴性菌的分布情况及耐药性特点,为规范抗生素的合理使用提供依据。方法选用法国梅里埃公司生产的ID 32GD试剂盒,应用ATB Expression全自动分析系统对细菌进行鉴定与药敏试验。结果2011～2015年在我院呼吸内科常见的病原菌分别为肺炎克雷伯菌（31．0％）、大肠埃希菌（21．2％）、铜绿假单胞菌（23．2％）与鲍曼不动杆菌（14．8％）。肺炎克雷伯菌对碳青霉烯类的耐药率几乎为0,对单纯的头孢菌素耐药率相对较高,但对含β‐内酰胺酶抑制剂的头孢菌素类以及阿米卡星有较强的抗菌活性;大肠埃希菌对哌拉西林耐药率高,但对含酶青霉素类有较强的抗菌活性,对碳青霉烯类的耐药率连续5年来均为零;铜绿假单胞菌对磺胺类耐药性较高,对头孢三代、头孢四代及含酶青霉素类与含酶头孢类均有较强的抗菌活性,但敏感性呈逐年下降趋势;鲍曼不动杆菌的耐药率呈逐年上升趋势,仅对含酶的头孢哌酮／舒巴坦敏感（约80％）。结论医院呼吸内科革兰阴性菌仍以肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌为主。鲍曼不动杆菌耐药性逐年增长的问题已日趋严重,相关部门应定期监测细菌耐药趋势,指导临床合理用药,特别是合理应用抗生素以避免细菌耐药及减少细菌耐药性的发生。     

临床分离肺炎克雷伯菌Ⅰ类整合子的结构与功能

为了探索细菌多重耐药性的产生和播散的分子机制, 文章对2002～2007年间179株临床分离的肺炎克雷伯菌进行耐药性、I类整合子可变区基因盒结构以及基因盒携带的耐药性基因进行分段克隆和耐药性功能测定。结果显示：65.9%(118/179)的肺炎克雷伯菌表现出对至少两种以上的抗生素(主要为β-内酰胺类、氨基糖苷类和喹诺酮类抗菌药物)的耐药性; 36.3%(65/179)的菌株检出单条或者双条I类整合子基因盒条带; 对整合子阳性组与阴性组的耐药率进行比较发现, 除氨基糖苷类、喹诺酮类和复方新诺明等药物的耐药性存在显著性差异(P<0.01)外, 其余药物的差异不显著; 共发现15种耐药基因构成形式的整合子基因盒, 其中以dfrA17-aadA5最为多见, 实验证明整合子可由接合转移耐药性质粒携带; 对整合子基因盒(dhfr17-orfF-aadA2)分段克隆的耐药性功能研究发现, 3个克隆重组子(pET28a-dhfr17、pET28a-dhfr17-orfF和pET28a-dhfr17-orfF-aadA2)对复方新诺明的抗性(MIC值)均为256 µg/mL, 重组子pET28a-dhfr17-orfF与重组子pET28a-dhfr17对链霉素的抗性无明显区别, 和受体菌一样MIC值均为8 µg/mL, 而pET28a-dhfr17-orfF-aadA2对链霉素的抗性则明显提高, MIC值为256 µg/mL。结果表明, I类整合子在肺炎克雷伯菌中较常见, 携带氨基糖苷类和甲氧苄啶类的耐药基因盒在数量上占优势, 且整合子携带的耐药基因具有耐药性功能, 位于可水平转移耐药性质粒的耐药性基因相关的整合子对病原菌耐药性播散具有重要意义。 目的基因     

Serological investigation of the fish pathogen Edwardsiella ictaluri, cause of enteric septicemia of catfish.

The serological relationships among 32 isolates of Edwardsiella ictaluri obtained from fish were studied. The strains were extremely homogeneous in protein and lipopolysaccharide preparations as observed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. Only minor variations were observed in the structural O-side chain subunits in three isolates; however, such variation did not preclude antigenic recognition by two E. ictaluri antisera in either microagglutination or Western blot immunoassays. The antigenic homogeneity of E. ictaluri was further demonstrated by microagglutination assays with both formalin-killed and heat inactivated cellular antigens. The minimal degree of antigenic variability observed suggested that most isolates of E. ictaluri compose a single antigenic serotype.     

Characterization of the rrn operons in the channel catfish pathogen Edwardsiella ictaluri

Aims:  To advance diagnostics and phylogenetics of Edwardsiella ictaluri by sequencing and characterizing its rrn operons.
Methods and results:  The Edw. ictaluri rrn operons were identified from a 5–7 kbp insert lambda library and from Edw. ictaluri fosmid clones. We present the complete sequences and analysis of all eight Edw. ictaluri rrn operons and unique regions located upstream and downstream. Two rrn operons were located in tandem with 169 bp separating them, which is apparently a conserved feature between Edw. ictaluri and Edwardsiella tarda. I- Ceu I enzyme digestion of Edw. ictaluri genomic DNA and analysis by pulsed field gel electrophoresis indicated that rrn operon number and chromosomal locations are conserved within the species Edw. ictaluri .
Conclusions:  The rrn operons of Edw. ictaluri have similar structure and flanking regions compared with other members of the family Enterobacteriaceae ; however, the presence of eight copies of the rrn operon makes Edw. ictaluri unique within the family .
Significance and impact of the study:  This research clarifies previous phylogenetic analyses of Edw. ictaluri and provides support for the Edw. ictaluri genome sequencing project. In addition, we identified a unique feature of two rrn operons that shows potential for the development of a diagnostic PCR method.     

HPLC fractionation and pharmacological assessment of green tea seed saponins for antimicrobial,anti-angiogenic and hemolytic activities

Herbal medicinal products have proven to be safe, economical and effective alternatives to synthetic chemical pharmaceuticals. The green tea plant (Camellia sinensis) is of profound medicinal value due to the presence of potent bioactive constituents. The purpose of the present work is to investigate saponins from green tea seeds for potential use as anti-angiogenic, antimicrobial, and hemolytic agents. Green tea seed saponins were separated into six fractions by reverse phase HPLC. The presence of three aglycone chains in the saponins of each fraction was confirmed by acid hydrolysis. Anti-angiogenic activity was evaluated using saponin fractions at concentrations in the range of 2.5 ~ 25 μg/mL. Antimicrobial activity was evaluated by thin-layer chromatography (TLC) using E. coli; S. mutans, a zoonotic Salmonella species and the fungal strain, A. niger. Saponin fractions were more potent against E. coli (gram negative bacteria) than against S. mutans (gram positive bacteria) and strongly inhibited six strains of zoonotic Salmonella. Green tea saponins also showed potent anti-angiogenic effects. All saponin fractions exhibited hemolytic activity. Our results confirm that green tea saponins have antimicrobial, anti-angiogenic, and hemolytic activities; indicating their potential as natural pharmaceutical products.