为了从红树林植物根际土壤中分离出具有广谱抑菌作用的海洋细菌,试验以大肠杆菌、金黄色葡萄球菌、沙门氏菌、链球菌4种常见致病菌为指示菌,采用M10和P3培养基从红树植物根际土壤中分离细菌,以固体琼脂打孔法对分离的细菌进行抑菌活性筛选,并提取分离菌株的基因组DNA,PCR扩增16S rDNA基因并测序,同时采用邻接法构建系统发育树.结果表明:从红树植物土壤中分离得到654株细菌,其中107株细菌具有抑菌活性,占分离菌株的16.4％.菌株M36对大肠杆菌和链球菌抑菌活性最强,抑菌圈直径分别为13 mm、16 mm;菌株P19和P25对金黄色葡萄球的抑菌活性最强,抑菌圈直径均为16 mm;菌株P83对沙门氏菌抑菌活性最强,抑菌圈直径为13 mm;菌株M36和P31的抑菌谱最广.综合两种培养基的培养结果有抑菌活性的菌株被分成4个属,分别是肠杆菌属、芽孢杆菌属、葡萄球菌属、伯克霍尔德氏菌属,其中肠杆菌属为优势菌属,其次为芽孢杆菌属,占比分别为69.2％和15.9％.说明红树植物根际土壤中存在具有广谱抑菌活性的细菌.

Introduction:The widespread use of antibiotics in animal agriculture has increased the resistance of Escherichia coli, and pathogenic E. coli often harbor complex virulence factors. Antimicrobial resistance in pathogenic bacteria can cause public health problems. Correlation analyses of the resistance, virulence, and serotype data from the pathogenic bacteria found on farms and in the surrounding environment can thus provide extremely valuable data to help improve public health management.Methods:In this investigation, we have assessed the drug resistance and virulence genes as well as the molecular typing characteristics of 30 E. coli strains isolated from duck farms in the Zhanjiang area of China. Polymerase chain reaction was used to detect the drug resistance and virulence genes as well as serotypes, and whole-genome sequencing was used to analyze the multilocus sequence typing.Results:The detection rates for the oqxA resistance gene and fimC virulence gene were highest (93.3%, respectively). There were no correlations between the drug resistance and virulence gene numbers in the same strain. The epidemic serotype was O81 (5/24), ST3856 was an epidemic sequence type, and strains I-9 and III-6 carried 11 virulence genes. The E. coli strains from the duck farms in the Zhanjiang area were thus found to have a broad drug resistance spectrum, various virulence genes, complex serotypes, and certain pathogenicity and genetic relationship.Discussion:Monitoring the spread of pathogenic bacteria and the provision of guidance regarding the use of antibiotics in the livestock and poultry industries will be required in the future in the Zhanjiang area.

[目的]了解常见致病菌产生耐药性后耐药基因和毒力基因的变化.[方法]采用微量肉汤二倍稀释法测定21种临床常见抗生素对大肠杆菌、金黄色葡萄球菌等5种标准菌株的最小抑菌浓度,选取敏感药物进行体外诱导耐药试验,采用PCR法检测耐药基因和毒力基因.[结果]5个标准菌株均对氨基糖苷类药物敏感;大肠杆菌对四环素类药物敏感;鼠伤寒沙门氏菌、多杀性巴氏杆菌和肺炎链球菌对喹诺酮类药物敏感;金黄色葡萄球菌对克林霉素敏感.与标准菌株相比,氨基糖苷类和四环素类耐药菌株中均检测出新出现的相关耐药基因;喹诺酮类耐药基因parE在多杀性巴氏杆菌的标准菌株和耐诺氟沙星菌株中检测出,gyrB基因仅在肺炎链球菌的标准菌株和耐氧氟沙星菌株中检测出.在大肠杆菌中均检测出CS31A毒力基因,此外标准菌株中检测出fimH基因,耐多西环素菌株中检测出afa基因;多杀性巴氏杆菌标准菌株未检测出毒力基因,耐药菌株中均检测出oma87和tbpA基因;而鼠伤寒沙门氏菌、金黄色葡萄球菌和肺炎链球菌仅在标准菌株中检测出毒力基因.[结论]说明氨基糖苷类抗生素更容易诱导菌株产生耐药基因,而毒力基因与耐药基因无明显的相关性.细菌获得耐药性后,其毒力基因变化因菌株不同存在差异.

Florfenicol is used worldwide for its low side effects and strong bactericidal effect. Florfenicol is physicochemically stable and can persist in natural water bodies and affect water denitrification. Indoor aquatic microcosm models were constructed and water samples were collected at different florfenicol concentrations (0.1, 1, 10, and 100 mg/L) on days 0, 7, 30, and 60 to extract the microbial genome DNA and determine the water properties. qPCR and amplicon sequencing were used to study the dynamic changes of nirS gene and nirS-type denitrifying communities structure, diversity and abundance, respectively. The results showed that higher florfenicol concentrations caused accumulation of nitrate and ammonium nitrogen in water. Florfenicol stress caused orders of magnitude changes in nirS gene abundance, showing a trend of increasing first and then decreasing. 100 mg/L florfenicol addition led to a sustained increase of nirS gene abundance in water bodies. The florfenicol addition altered denitrifying community structure and suppressed the richness and diversity index of denitrifying bacteria in water body. Over time, the richness and diversity index gradually recovered. Proteobacteria was always the dominant denitrifying phylum in water. The relative abundance of Pseudomonas and beta proteobacterium showed obvious positive correlation with nirS gene abundance and were the dominant genera under florfenicol stress. Our study provided a scientific basis for the rational use of florfenicol in aquaculture to maintain a healthy and stable microecological environment, and also provided a preliminary understanding of the response characteristics of water denitrifying microorganisms to florfenicol exposure.

从土壤中分离得到10株解磷菌,将菌株接种至LB液体培养基中,30℃培养24 h作为种子液.在无机磷液体培养基中加入氟苯尼考,使培养基含药浓度分别为0、0.1、1.0、10.0、50.0 μg/mL.按照1%的接种量接种种子液,30℃培养5 d,每24 h取2 mL培养液,利用钼锑抗分光光度法测定培养液中速效磷含量.结果表明,10株解磷菌在50.0 μg/mL药物浓度下,培养基中速效磷含量明显低于其他药物浓度组;大部分菌株在药物浓度0～50.0 μg/mL菌液中细菌数量变化不明显.说明氟苯尼考对解磷菌的解磷能力产生了抑制作用,但与细菌的生长繁殖并无明显相关性.

Florfenicol is one of the most widely used antibiotics in aquaculture and veterinary clinics because of its low side effects and strong bactericidal effect. A total of 45~60% of florfenicol is not absorbed by the animal body and accumulates in the aquatic environment through a variety of pathways, which affects denitrification. Indoor aquatic microcosm models were constructed and sediment samples were collected at different florfenicol concentrations (0.1, 1, 10, and 100 mg/L) on days 0, 7, 30, and 60 to extract the microbial genome DNA and determine the water properties. qPCR and amplicon sequencing were used to study the dynamic changes in the nirS gene and nirS-type denitrification community structure, diversity, and abundance, respectively. The results showed that high florfenicol stress influenced the sediment's physicochemical properties, reducing conductivity, alkaline dissolved nitrogen, and organic matter content. In addition, the abundance of nirS, a functional denitrification gene, increased obviously with increased florfenicol concentrations but decreased the diversity of nirS-type denitrification microorganisms. Proteobacteria was the dominant denitrifying phylum in the sediment. Our study provides a scientific basis for the rational use of florfenicol in aquaculture to maintain a healthy and stable microecological environment and also provides a preliminary understanding of the response characteristics of water denitrifying microorganisms to florfenicol exposure.

In order to explore the impact of antibiotics (enrofloxacin) on microbial community in aquatic environment, an indoor aquatic ecological model was built, and different concentrations of enrofloxacin (0.05, 0.5, 5, and 50 mg/L) were added in the aquatic ecological model. In addition, the water and sediment samples were collected on the 0, 7, 30, and 60 days, and the changes in microbial community were studied through 16S rDNA high-throughput sequencing. The results showed that when the concentration of enrofloxacin was 50 mg/L, the relative abundance of Actinomycetes was increased. In the water, the bacterial richness and diversity communities first decreased and then gradually recovered with the passage of time; On the 7th day, the diversity and richness index of species in the treatment groups with enrofloxacin at 5 and 50 mg/L decreased to the lowest; On the 30th day, the diversity and richness index of species began to rise; On the 60th day, the diversity index and richness index of water species began to increase, while the diversity index and richness index of sediment species decreased. In conclusion, the addition of enrofloxacin negatively affected the microbial community structure in an indoor aquatic ecological model, 50 mg/L enrofloxacin could increase the relative abundance of Actinomycetes, and decrease the diversity and richness index of water and sediment.

This study evaluates the effects of a broad-spectrum antibiotic (florfenicol) on antibiotic resistance genes (ARGs) and bacterial community structure in aquatic environments. We constructed an indoor aquatic microcosm model, adding different concentrations of florfenicol (0.1, 1, 10, 100 mg L−1), and water and sediment samples were collected after 0, 7, 30, and 60 days. qPCR and 16S rDNA amplicon sequencing were used to study the changes in the ARGs and bacterial community structure of the collected samples. The results show that the inclusion of florfenicol resulted in an increased abundance of the floR and optrA genes. Adding 100 mg L−1 florfenicol to the water increased the abundance of optrA gene copies with the maximum on the Day 7, and increased the abundance of floR gene copies with the maximum on Day 30. Adding 100 mg L−1 florfenicol to the sediment increased the abundance of floR and optrA genes by one order of magnitude on Day 60. Meanwhile, the average number of operational taxonomic units (OTUs) in the water samples was 257, and the average number of OTUs in sediment samples was 823. The bacterial community diversity and richness in sediments were higher than those in water. The difference between the maximal and minimal values of the Shannon diversity index in the water and sediment samples was 4.36 and 1.95, respectively. The effect of florfenicol on the bacterial community structure in water was much higher than that in sediment. At 30 days, the diversity index and richness index of the florfenicol treatment groups with 1 and 10 mg L−1 concentrations began to increase; at 60 days, the diversity and richness indices of the 100 mg L−1 florfenicol treatment group began to increase. The samples at the same sampling time in the sediments clustered closer together. The results of this study provide a scientific basis for guiding the rational use of florfenicol in aquaculture, maintaining a healthy and stable microecological environment in aquaculture, and provide theoretical data for environmental ecological risk assessment and safety management caused by microbial resistance under the abuse of florfenicol.

研究石榴皮、花椒、大青叶等9种中草药对沙门菌和金黄色葡萄球菌的体外抑菌作用.采用水煎煮法分别提取9种不同中草药的有效成分;采用二倍稀释法、平板培养法分别检测中草药对沙门菌和金黄色葡萄球菌的最低抑菌浓度(MIC)和最小杀菌浓度(MBC),筛选出抑菌功效良好的5味中草药,再分别对2种细菌进行联合用药,测定中草药联用的体外抑菌功效.结果表明,花椒、黄连、石榴皮、诃子、龙胆草等单味药具有较好的体外抑菌功效,在9种中草药中抑菌作用最佳的是黄连,其对沙门菌和金黄色葡萄球菌的MIC值分别为1.625 mg/mL和3.125 mg/mL,MBC值分别为100.000 mg/mL和200.000 mg/mL.对金黄色葡萄球菌,中草药间有拮抗作用的组合有黄连+花椒、黄连+诃子、石榴皮+龙胆草,其他中草药联合时均为无关作用;对沙门菌,中草药间有拮抗作用的组合有黄连+花椒、诃子+龙胆草、石榴皮+诃子、黄连+诃子、石榴皮+龙胆草,中草药间有相加作用的组合是花椒+石榴皮,其MIC值为3.125 mg/mL,FIC值为0.75,其他中草药联合时均为无关作用.结果提示,单独用药对细菌具有良好抑菌功效的中草药,联合用药时抑菌作用未必会增强.

研究苏木、黄连、石榴皮等20味中药对大肠杆菌的体外抑菌作用.采用水提法制备20味中药的提取物,通过试管二倍稀释法测定20味中药水提取物对大肠杆菌的最低抑菌浓度(MIC)和最小杀菌浓度(MBC);再将20味中药组成药对,进行联合抑菌,测定药对的MIC和MBC,计算其部分抑菌浓度指数(FIC).结果表明,20味中药中,黄连、苏木抑菌效果最佳,MIC均为6.250 mg/mL,茯苓、山楂、火麻仁抑菌效果最差,其MIC和MBC均大于400.000 mg/mL.联合抑菌的28组药对中,苏木与石榴皮、石榴皮与黄柏、石榴皮与黄芩、地榆与苏木、地榆与乌梅、地榆与花椒、地榆与黄柏、火麻仁与旋覆花、旋覆花与山楂等9组药对均呈现协同作用,石榴皮与黄连、石榴皮与乌梅均呈现拮抗作用,其他药对呈现相加或无关作用.说明对大肠杆菌抑菌作用强的单味中药与其他中药联合,其抑菌效果未必会增强.