细菌耐药性研究进展

近年来由于抗菌药物的不合理应用,从而导致细菌对抗菌药物的耐药性现象日趋严重。文章从细菌耐药机制、抗菌药物耐药性风险评估方法及细菌耐药性消除3个方面进行分析和总结,以期能为解决细菌耐药性问题和研发新型绿色抗菌药物提供思路。     

水产动物源细菌耐药性与预防控制

细菌耐药性是指细菌对于抗菌药物作用的耐受性。细菌耐药性的形成,是细菌进化发展的结果,是细菌适应性的表现,但它却给养殖业的发展和人类的健康带来威胁。本文首先综述了水产动物源细菌耐药性研究现状,主要包括质粒介导的水产动物源细菌耐药性、细菌耐药基因、细菌耐药性遗传学机制等;其次,从病原菌耐药性会导致水产疾病流行爆发和水产细菌耐药性对人类公共卫生安全的影响两方面,分析了水产动物源细菌耐药性的危害;最后,从合理、规范用药,加快水产疫苗和新型抗菌药物替代品的研制与应用,加强水产动物致病菌耐药性监测等方面,提出了预防和控制水产动物源细菌耐药性的对策和措施。     

鱼类铁调素生物学功能与体外表达的研究进展

<正>近年来，细菌性疾病频发，抗生素不规范使用导致细菌耐药性问题日益严重^[1]。研发新的抗菌药物，替代抗生素是解决细菌耐药性问题的一个方向。抗菌肽是具有抗菌作用的小分子多肽，相比于抗生素，不易产生耐药性，可作为抗生素耐药细菌治疗的潜在药物^[2-3]。其中鱼类铁调素(Hepcidin)具有广谱的抗菌活性，抗菌效果明显，已成为当前抗菌肽研究的热点。     

L-半胱氨酸提高嗜水气单胞菌对氟苯尼考的敏感性

抗菌药物的应用能对细菌感染性疾病进行有效地控制和治疗,但水产动物养殖中滥用和误用抗菌药物导致耐药细菌的产生、养殖水环境污染和养殖动物药物残留等一系列问题,应对和解决抗菌药物在水产动物养殖中的滥用问题已经刻不容缓。本研究通过抗菌药物杀菌、组织细菌清除、细菌攻毒、鱼体存活率统计和药物敏感性检测等方法,探究外源L-半胱氨酸添加对氟苯尼考杀菌作用的影响。体外杀菌结果显示,4.00 mmol/LL-半胱氨酸可使20.00～200.00μg/mL氟苯尼考对嗜水气单胞菌的杀菌效率提高1.9～11.1倍,其联合用药的杀菌作用具有剂量和时间依赖效应。组织细菌清除结果显示,相比单独使用氟苯尼考,L-半胱氨酸与氟苯尼考合剂对鱼体肝脏、脾脏和肾脏组织中嗜水气单胞菌的清除能力提高4～16倍。细菌攻毒后,采用注射和口服L-半胱氨酸或/和氟苯尼考进行治疗,结果发现L-半胱氨酸与氟苯尼考合剂可大幅提高黄河鲤对嗜水气单胞菌的抗感染能力。同时,单独添加L-半胱氨酸也能促进组织细菌的清除和鱼体的抗感染能力。细菌MIC测定结果显示,添加1.00～4.00 mmol/L L-半胱氨酸使嗜水气单胞菌对氟苯尼考的敏感性提高2～4倍。由此推测,L-半胱氨酸可能通过增强嗜水气单胞菌对氟苯尼考的敏感性,从而提高抗菌药物对鱼体细菌感染的防治效果。研究表明,L-半胱氨酸作为氟苯尼考的增效剂不仅可提高抗菌药物的防治效果,而且可大幅降低抗菌药物的使用量,对嗜水气单胞菌的防控具有一定的实际应用价值。     

优马林与福尔马林、硫酸铜、硫酸锌的比较及其在水产养殖中的应用

1优马林的特点及其与几种常用药物的比较优马林是一种具有强力协同作用的环保型消毒、杀菌、杀虫剂,其杀虫抗菌机理是能与寄生虫、细菌性蛋白起反应,蛋白质不断氧化分解,最后导致虫体及细菌细胞死亡。本品具有以下优点:1)优异的杀虫功能:迄今为止最好的杀车轮虫、斜管虫的药物;2)良好的防治细菌性疾病(如白鳃、白肝、烂鳃病)的功效:可快速抑制微生物生长,导致微生物的死亡,不必再用或少用杀菌药;3)药效持续时间长:防病、治病药效持续时间长,可较长时间抑制细菌、杀死寄生虫;4)极易生物降解:在稀释浓度范围内降解为无毒物质,对环境无害;5)适…     

优马林在水产养殖中的应用（上）

一、优马林的特点以及与几种常用药物的比较优马林是一种具有强力协同作用环保型消毒、杀菌、杀虫剂,其杀虫抗菌机理是能与寄生虫、细菌性蛋白起反应,蛋白质不断氧化分解,最后导致虫体及细菌细胞死亡。本品具有以下优点:①优异的杀虫功能:是迄今为止最好的杀车轮虫、斜管虫的药物;②良好的防治细菌性疾病(如白鳃、白肝、烂鳃病)的功效:可快速抑制微生物生长,导致微生物的死亡,不必再用或少用杀菌药;③药效持续时间长:防病、治病药效持续时间长,可较长时间抑制细菌、杀死寄生虫;④极易生物降解在稀释浓度范围内降解为无毒物质,对环境无害;⑤…     

鱼病防治实用技术(连载)

（6）喹诺酮类抗菌药物这是一类化学合成的抗菌药品,具有抗菌谱广（尤其对革兰氏阴性菌）、杀菌力强的特点,对耐抗生素的细菌也有杀菌作用。细菌对这类药不容易产生耐药性。本类药物口服吸收快,半衰期较长。近年来在我国使用较多。国外使用最多的有以下几个品种：茶咬酸、供喷酸、此咯咯灾酸（或称吮咯酸）、氟嚷酸、氯甲喷、恩诺沙星（国外的商品名为百病消）、沙拉氟诺沙星等。它们之间有交叉耐药性,与土霉素之间也有交叉耐药性的可能。本类药物受水的硬度、pH以及二价阳离子影响较大,所以在海水中使用时需加大剂量。喷酸：为白色粉…     

水生动物药物残留的危害与控制

1 水产动物药物残留的危害1.1 耐药性反应 在水生动物饲料中长期添加促生长抗菌素或生产中滥用药物会导致水生动物体内的细菌产生耐药性。研究表明,随着抗菌药物的不断应用,细菌中耐药菌株的数量在不断地增加。这些细菌的耐药性可以通过耐药因子(R-质粒)在人群中细菌、动物群中细菌和生态系统中细菌间互相传递,导致致病菌产生耐药     

几种抗菌药物的作用和用法介绍

1.呋喃类药物呋喃类药物具有广谱抗菌作用,在体外对革兰氏阳性及阴性球菌、杆菌都有抑制作用,20～25微克/毫升能杀菌,5～10微克/毫升能干扰细菌体内氧化还原酶系统,使细菌代谢紊乱,特点是不易产生耐药性。对变形杆菌和假单胞菌的抗菌力较差。     

冬凌草甲素对嗜水气单胞菌体外抑菌效果及作用机制

通过测定冬凌草甲素对嗜水气单胞菌的最低抑菌浓度(minimum inhibitory concentration, MIC)、最低杀菌浓度(minimum bactericidal concentration, MBC)和对细菌生长的影响,及其对菌体形态、电导率、乳酸脱氢酶(lactate dehydrogenase, LDH)活性、蛋白质和DNA的影响,探究冬凌草甲素对耐药性嗜水气单胞菌的体外抑菌作用及其机制。结果显示,冬凌草甲素对嗜水气单胞菌具有明显抑制作用,MIC为256μg/mL,MBC为512μg/mL。当2MIC浓度的冬凌草甲素作用于嗜水气单胞菌CW株8、16 h后,菌体变形,细胞壁和细胞膜分离,细胞质空化。与对照组相比,药物组菌悬液的电导率显著升高,并且在冬凌草甲素作用6 h后达到5.66%。冬凌草甲素作用8 h后嗜水气单胞菌的LDH活性降低了20.8%,可溶性蛋白含量显著降低。DAPI染色结果显示,药物组荧光密度和强度均较对照组弱。DNA外渗量结果显示,较对照组上升了(29.32±1.02) mg/L。研究表明,冬凌草甲素对嗜水气单胞菌具有较强的抗菌作用,主要通过增加细胞膜的通透性,干扰蛋白质代谢进而抑制细菌的生长繁殖。     

罗氏沼虾胞质锰超氧化物歧化酶的功能

为了解超氧化物歧化酶 (SOD)分子生物学特性和免疫功能，实验克隆了罗氏沼虾胞质锰SOD基因 (MrcMnSOD)，制备多克隆抗体，并分析在嗜水气单胞菌感染下该基因的表达模式。结果显示，嗜水气单胞菌感染可显著诱导MrcMnSOD在转录水平和蛋白质水平进行高表达。为探究MrcMnSOD参与免疫应答的机制，进一步的抑菌实验表明，该蛋白质可显著抑制3种革兰氏阴性细菌 (大肠杆菌、副溶血性弧菌、嗜水气单胞菌)和2种革兰氏阳性细菌 (无乳链球菌、金黄色葡萄球菌)的生长，且抑制作用与蛋白质浓度的关系不显著。研究表明，MrcMnSOD可能作为一种免疫相关分子参与免疫应答反应。本研究初步探讨了MrcMnSOD的免疫生物学功能，旨在为深入研究罗氏沼虾SOD的功能奠定相关基础。     

Bioassay‐guided isolation and identification of active compounds from Macleaya microcarpa (Maxim) Fedde against fish pathogenic bacteria

Four alkaloids (Sanguinarine, 6‐Methoxyl‐dihydro‐chelerythrine, Cryptopine and β‐Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay‐guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram‐negative strains than other three alkaloids, with MIC values of 12.5 mg L^?1 for A. hydrophila and 50 mg L^?1 to other pathogenic bacteria. Followed by 6‐methoxyl‐dihydro‐chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L^?1 for A. hydrophila, 100 mg L^?1 for V. harveyi, and 125 mg L^?1 for both V. anguillarum and A. salmonicida. Cryptopine and β‐allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L^?1 for A. hydrophila and 200 mg L^?1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.     

山瑞鳖细菌性败血症病原菌的分离鉴定及其毒力基因检测

查明广西南宁、贵港和桂平养殖山瑞鳖细菌性败血症的病原菌及其6种毒力基因的携带情况,为有效防控山瑞鳖细菌性败血症提供参考。本研究以常规方法从患病山瑞鳖的心脏和肝脏取样、分离细菌,人工感染方法确定分离菌株的致病性,细菌鉴定采用API 20NE生化鉴定和16S rRNA分子鉴定相结合的方法进行,PCR扩增法对菌株的溶血素基因(hemolysin gene,hly)、气溶素基因(aerolysin gene,Aer)、细胞兴奋性肠毒素基因(cytotonic enterotoxin gene,Alt)、细胞毒性肠毒素基因(cytotoxic enterotoxin gene,Act)、黏附素基因(major adhesin gene,ahal)和丝氨酸蛋白酶基因(serine protease gene,ahp)6种毒力基因进行检测。结果显示,从患病山瑞鳖心脏和肝脏中共分离到4株优势菌SRB125、SRB142、SRB143和SRB345,对健康山瑞鳖的平均致死率为97.50%~100.00%,是引起山瑞鳖细菌性败血症的病原菌;生化和分子鉴定结果显示,4株分离菌均为嗜水气单胞菌(Aeromonas hydrophila),与A.hydrophila L3-5(KP716701)菌株的亲缘关系最近,同源相似性均达到99.9%;6种毒力基因共包含2种毒力基因型,在4株菌株中的分布为hly~+Aer~+Alt~+Act~+ahal~+ahp~+和hly~+Aer~+Alt~+Act~+ahal~+ahp~–各2株,来源于南宁的SRB143和桂平的SRB345菌株均缺失ahp基因。     

Ethnobotanical Uses and Antimicrobial Properties of Plants in Small‐Scale Tropical Fish Farms: The Case of Indonesian Fish Farmers in Java (Indonesia)

Among the most common plants used by fish farmers in Java, 18 were selected for deeper ethnobotanical and laboratory investigation into their antimicrobial activity. These plants are mainly used against fish diseases, but with no precise therapeutic indication, leading to low specificity of use. Leaves, which were the most commonly used part of plants, were generally placed directly in water or less frequently added to the feed. Disk diffusion tests showed that 15 plants of the 18 (83%) displayed varying degrees of antibacterial activity and that Streptococcus agalactiae was more sensitive than Aeromonas hydrophila. Crude ethanolic extracts (EE) were more active than water macerates (WM), and minimum inhibitory concentrations (MICs) ranged from 12.5 to 25 mg/mL. Only the EE of Piper betle had an inhibitory effect against A. hydrophila. No MIC was found for any of the WM studied here. Furthermore, both the EE and, to a lesser extent, the WM of active plants were able to reduce bacterial growth by more than 99%, even at doses below their MICs. These results suggest that these plant extracts have a potential for eco‐friendly prevention of bacterial fish diseases.     

A Study on the antibacterial mechanism of thymol against Aeromonas hydrophila in vitro

As a zoonotic pathogen, Aeromonas hydrophila (A. hydrophila) not only has important economic significance in aquaculture but also plays an important role in global public health. With the abuse of antibiotics, the drug resistance of A. hydrophila has greatly increased, causing a poor therapeutic effect. It is particularly important to develop antibiotics alternatives against resistant bacteria. Thymol, a natural monoterpene phenolic compound, has been reported to have a variety of pharmacological effects. In this study, the effects of thymol on A. hydrophila were evaluated. The MIC and MBC of thymol to A. hydrophila were 128 μg/mL and 256 μg/mL, respectively. The results of the electron microscope showed that the bacterial cells contracted and deformed, the cell membrane was destroyed, and the cytoplasm was lost when treated with 2MIC thymol. Compared with the control group, the conductivity and the amount of DNA leakage of bacteria treated with 2MIC thymol increased by 2.45% and 66.4 μg/mL, respectively. In addition, LDH activity decreased by 25.93% after 2MIC thymol treatment. DAPI staining results showed that the intensity and quantity of fluorescence were significantly decreased in the drug treatment group. In summary, the antibacterial mechanism of thymol was to destroy the integrity of the cell membrane, make intracellular material exudate, and eventually lead to the death of bacteria. Thymol has the potential to be an antibiotics alternative against resistant bacteria.

     

Aerobic Bacterial Flora of Common Carp (Cyprinus carpio L.) Cultured in Earthen Ponds in Saudi Arabia

ABSTRACT

Quantitative and qualitative estimation of bacterial flora present in pond water, sediments, gills, and intestine of healthy common carp Cyprinus carpio cultured in Saudi Arabia were performed and identified to species level where possible. Mean total viable bacterial counts in pond water ranged from 1.2?±?2.9?×?10⁴ to 2.5?±?3.5?×?10⁵ cfu/mL; in sediments, 9.3?±?2.1?×?10⁷ to 2.7?±?3.5?×?10⁹ cfu/g; in gills filaments, 4.3?±?2.9?×?10⁶ to 1.6?±?3.9?×?10⁷ cfu/g; and in intestine, 8.7?±?4.1?×?10⁹ to 5.4?±?3.2?×?10¹⁰ cfu/g. Gram-negative rod-shaped bacteria dominated (76%) the populations. In total, 12 bacterial genera and 15 species were identified. Pond water and sediment bacteria had the reflection on bacterial composition of gills and intestine of carp. Intestinal bacteria showed more diversification in contrast to gill bacteria. Aeromonas hydrophila, Shewanella putrefaciens, Vibrio cholerae, Corynebacterium urealyticum, Vibrio vulnificus, Vibrio sp., and Staphylococcus sp. were the common bacteria in all the populations. In pond water and carp intestine, A. hydrophila, S. putrefaciens, V. cholerae, and C. urealyticum were the most dominant bacteria (prevalence ≥ 10%) where pond sediments and the carp gills experienced with more one dominant bacterium V. vulnificus. Only the A. hydrophila covered one fourth (25%) of the total bacterial populations.     

Analysis of virulence and immunogenic factors in Aeromonas hydrophila: Towards the development of live vaccines

Aeromonas hydrophila, a bacterium that is widespread in aquatic environments, is responsible for causing haemorrhagic disease in both aquatic and terrestrial species. With the purpose of developing a live vaccine, herein we have investigated nine strains of A. hydrophila (Ah-01 to Ah-09) isolated from diseased fish. A study of virulence factors that contribute to pathogenicity and immunogenicity in the host Cyprinus carpio suggests that the presence of β-hly, act and fla genes contribute to pathogenesis: strains Ah-01, Ah-02 and Ah-03 (β-hly⁺/act⁺/fla⁺ genotype) were highly pathogenic to C. carpio, whereas Ah-05 and Ah-06 (β-hly⁻/act⁻/fla⁻ genotype) showed weak pathogenicity. Accordingly, Ah-02 and Ah-03 were selected to prepare inactivated vaccines, whereas Ah-05 and Ah-06 were chosen as live vaccines. Ah-06 live vaccine was found to have the best protective efficacy, with a protective rate of about 85%, whereas rates of other vaccines were significantly lower, in the range 37%–59%. In addition, DNA vaccines based on genes altA, aha and omp showed immune protection rates of 25%, 37.5% and 75%, respectively. Our data demonstrate that the β-hly⁻/act⁻/fla⁻/altA⁺/aha⁺/omp⁺ genotype has weak pathogenicity and high immunogenicity, and provide a simple and effective way to screen for live A. hydrophila vaccines.     

In vitro antibacterial activity of four nano‐encapsulated herbal essential oils against three bacterial fish pathogens

Bacterial pathogens causing diseases in fishes have broad host ranges, and may cause high mortality or ongoing chronic infections. Pathogen treatments using antibiotics are becoming limited due to increasing concerns for the development of resistance and environmental dissemination of bacteria harboring resistance genes. This study aimed to determine whether nano‐emulsions of selected plant‐derived essential oils (Origanum vulgare, Eucalyptus globulus, Melaleuca alternifolia and Lavendula angustifolia) were bacteriostatic or bactericidal to Aeromonas hydrophila, Streptococcus iniae and Photobacteriumdamselae subspecies damselae. All treatments showed antibacterial activity, and in almost all cases the activity of the nano‐emulsions was superior to their essential oil counterparts. Origanum vulgare (oregano) nano‐emulsion had the most effective antibacterial activity, with a minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 3.12 µg/ml against all three species; substantially better than tetracycline. The present study highlights the potential of new formulations of essential oils to enhance bacterial disease control in cultured fish.     

Screening of gastrointestinal bacteria of Indian major carps for a candidate probiotic species for aquaculture practices

Probotic selection could be detrimental for the success of aquaculture practices. While, in most of the cases emphasis is given to the production of antibacterial compounds, and in vitro inhibitory activities of a bacterium for choosing a probiotic strain, however, other criteria like non‐antibiotic resistant, non‐haemolytic nature and non‐pathogenic potentiality of probiotic strain are often overlooked. We have screened gastrointestinal bacteria isolated from all the three species of Indian major carps viz., Labeo rohita, Catla catla and Cirrhinus mrigala for a suitable probiotic. Bacteria like Aeromonas (Aeromonas species, A. hydrophila), Micrococcus, Corynebacterium, Plesiomonas, Bacillus (Bacillus species, B. subtilis) and Pseudomonas (Pseudomonas species, P. aeruginosa) exhibited antibacterial activities against different pathogens. Among these, B. subtilis was found to grow at a wider range of temperatures, pH and salt concentrations and was non‐haemolytic, non‐antibiotic resistant, non‐pathogenic and inhibited all the tested primary and secondary pathogens. Dietary supplementation of the bacteria at 10⁸ CFU g^?1 feed also enhanced the growth of Indian major carp, L. rohita. Based upon these characteristics, we aimed to develop a suitable probiotic for aquaculture practices.