Isolation,Identification and Multidrug-resistant Analysis of the New Sequence Type ST989 Strain of Enterococcus faecium

Gram-positive cocci were isolated from half a month diseased lambs with a fatal infection in a sheep farm in Minhe county, Qinghai province.The clinical symptoms observed were drooping, reduced feed intake, emaciated and fever.The infected lambs died several hours after the disease been observed.Necropsy of two dying lambs revealed that for one lamb, the liver was enlarged and capsule of the liver dropped, and other organs had no obvious changes;For the other one, the organs had no obvious changes.After gram staining, gram-positive cocci were observed from all the samples.Partial Tuf gene sequences of these isolates were amplified and sequenced for species identification, BLASTn conparison result revealed that they had the highest sequence similarity with that of Enterococcus faecium (E.faecium) AUS0085 (99.8%), we thus primarily confirmed that these strains belonged to E.faecium.Antimicrobial susceptibility test indicated that these strains could be divided into two classes.Class 1 was resistant to β-lactams, aminoglycosides, macrolides, tetracyclines, quinolones, rifamycins, lincomycin and clindamycin;Susceptible to chloromycetin and vancomycin.Class 2 was resistant to β-lactams, aminoglycosides, tetracyclines, quinolones, rifamycins;Intermediate resistance to erythromycin and roxithromycin;But susceptible to clarithromycin, azithromycin, clindamycin, lincomycin, chloromycetin and vancomycin.Multilocus sequence typing (MLST) based on seven housekeeping genes for E.faecium revealed that E.faecium lby1 strain in class 1 and E.faecium lbg3 strain in class 2 were defined as a new sequence type ST989.Phylogenetic tree analysis revealed the representative strain of these isolates, E.faecium lbg3, was clustered together with E.faecium ST468.The pathogenicity test revealed that E.faecium lby1 and lbg3 strains both had pathogenicity.     

3种不同微生态制剂两两组合对池塘水质的影响

研究了3种微生态制剂枯草芽孢杆菌(A)、粪链球菌(B)、光合细菌(C)两两配伍,以105 CFU/ml的浓度按9∶1、4∶1、1∶1、1∶4、1∶9混合制成15种制剂,对池塘水质中COD、氨态氮、亚硝酸盐氮、硫化物的影响.结果表明,以1:4混合制成的枯草芽孢杆菌和光合细菌的混合制剂对COD的处理效果最好(P＜0.05),以4∶1混合制成的枯草芽孢杆菌和粪链球菌的混合制剂对氨态氮的处理效果最好,对亚硝酸盐氮处理效果最好的是以4∶1配制的粪链球菌和光合细菌的混合制剂(P＜0.05),对硫化物的处理效果最好是以9∶1混合制成的粪链球菌和光合细菌的混合制剂(P＜0.05).     

Acquired antimicrobial resistance in the intestinal microbiota of diverse cat populations

The aim of this study was to investigate the prevalence of acquired antimicrobial resistance in the resident intestinal microbiota of cats and to identify significant differences between various cat populations. Escherichia coli, Enterococcus faecalis, E. faecium and Streptococcus canis were isolated as faecal indicator bacteria from rectal swabs of 47 individually owned cats, 47 cattery cats and 18 hospitalised cats, and submitted through antimicrobial sensitivity tests. The results revealed that bacteria isolated from hospitalised and/or cattery cats were more frequently resistant than those from individually owned cats. E. coli isolates from hospitalised cats were particularly resistant to ampicillin, tetracycline and sulfonamide. Both enterococci and streptococci showed high resistance to tetracycline and in somewhat lesser extent to erythromycin and tylosin. Most E. faecium isolates were resistant to lincomycin and penicillin. One E. faecalis as well as one E. faecium isolate from hospitalised cats showed 'high-level resistance' (MIC > 500 microg/ml) against gentamicin, a commonly used antimicrobial agent in case of human enterococcal infections. The results of this research demonstrate that the extent of acquired antimicrobial resistance in the intestinal microbiota of cats depends on the social environment of the investigated population. It is obvious that the flora of healthy cats may act as a reservoir of resistance genes.     

粪肠球菌F71的增殖培养基优化研究

为达到增殖菌体的目的,对粪肠球菌F71进行了培养基优化正交试验。结果表明,最佳培养基的组成为:胰蛋白胨10 g/L,酵母粉10 g/L,葡萄糖10 g/L,无水氯化钙0.04 g/L,七水合硫酸镁0.019 2 g/L,磷酸氢二钾0.04 g/L,磷酸二氢钾0.04 g/L,磷酸氢钠0.4 g/L,氯化钠0.08 g/L,土豆汁50 g/L,碳酸钙1 g/L,乙酸钠10 g/L,碳酸铵2 g/L,乙酸铵1 g/L,半胱氨酸盐酸盐0.5 g/L,pH值为9,最适培养温度为35℃。用此培养基进行增殖,F71的最高活菌数达到2.78×108 cfu/mL,比基础培养基提高了54.6%。     

Enterococcus faecalis of human and poultry origin share virulence genes supporting the zoonotic potential of E. faecalis

Enterococcus faecalis is a major cause of nosocomial infections in humans and has been linked to severe extra‐intestinal infections in poultry. A zoonotic potential has been suggested and the aim of the present study was to investigate similarities in virulence gene profiles of E. faecalis originating from infections in humans and poultry respectively. A total of 106 isolates of E. faecalis [26 human clinical isolates, 60 poultry clinical isolates (including two small‐colony variants (SCVs) and 20 poultry cloacal isolates] were investigated for presence of seven virulence‐associated genes: ace, asa1, cylA, efaA, EF0591, esp and gelE. For each gene, the PCR‐amplification product was sequenced from one isolate in each group to explore intragenic variations between genes of human and poultry origin. Haemolytic and protease activities were assessed and isolates were assigned a sequence type (ST). Three of the seven genes investigated (ace, efaA and gelE) were present in all isolates. The asa1 was detected in 63/80 and 13/26 isolates of poultry and human origin respectively. For cylA, the numbers were 46/80 and 14/26 respectively. Among poultry isolates, esp and EF0591 were the least frequently observed genes (1/80 and 20/80 respectively); the prevalences among human isolates were 1/26 and 18/26 respectively. A high degree of similarity between genes in human and poultry isolates were confirmed by sequencing of amplification products. None of the cylA‐positive isolates demonstrated haemolytic activity, while the phenotypic expression of gelatinase varied. The ST16 was the only ST shared by human and poultry isolates. The SCV isolates did not show a unique virulence profile or phylogeny. In conclusion, regardless of the distinct phylogenetic background of most E. faecalis isolates of human and poultry origin, we found major similarities in virulence gene profile and gene sequences in isolates from the two sources, supporting the zoonotic risk associated with this organism.     

表达猪乳铁蛋白肽重组屎肠球菌对断奶仔猪生长性能的影响及其抗ETEC感染效果研究

本试验以表达猪乳铁蛋白肽的重组屎肠球菌（pNZ8112-PLFcin/Ef）饲喂断奶仔猪，研究其对仔猪生长性能的影响和抗产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）感染的效果。选取28日龄体重相近的健康断奶仔猪36头，随机分为3组（重组屎肠球菌组、空载体组和培养基组），每组3个重复，每个重复4头仔猪。重组屎肠球菌组和空载体组分别饲喂添加pNZ8112-PLFcin/Ef （6×10¹² CFU/kg）和pNZ8112/Ef （6×10¹² CFU/kg）的基础日粮，而培养基组饲喂含有相同体积的GM17液体培养基的基础日粮。试验期26 d。结果显示，与培养基组相比，重组屎肠球菌组断奶仔猪的平均日增重极显著提高（P<0.01）；料重比显著降低（P<0.05）；腹泻率明显降低，肠道菌群的均匀度和多样性指数均下降。为进一步探究表达猪乳铁蛋白肽的重组屎肠球菌对断奶仔猪抵抗ETEC感染的保护作用，在连续饲喂21 d后，每个重复中随机挑选1头体况相近的断奶仔猪灌服ETEC。结果发现，与培养基组相比，攻菌后重组屎肠球菌组断奶仔猪血清白细胞介素-2（IL-2）、免疫球蛋白G （IgG）含量、肠黏液中分泌型免疫球蛋白A （sIgA）水平均显著升高（P<0.05）；脾脏指数显著提高（P<0.05），但胸腺指数、肠段长度及重量则均无显著差异（P>0.05）。综上所述，表达猪乳铁蛋白肽的重组屎肠球菌能够起到促进断奶仔猪生长及抗ETEC感染的保护作用。     

Enterococcus faecium NCIMB 10415 supplementation improves the meat quality and antioxidant capacity of muscle of broilers

The present study was conducted to investigate the effects of Enterococcus faecium (E. faecium) on the meat quality and antioxidant capacity of muscle in broilers. A total of 600 Arbor Acre broiler chickens (1‐day‐old, male) were randomly divided into five treatments with six replicates (20 chickens per replicate) for each treatment. The five treatments were the control treatment (CON, basal diet), antibiotic treatment (ANT, basal diet supplemented with 0.1% chlortetracycline) and E. faecium‐supplemented treatments (LEF, MEF and HEF, basal diet supplemented with 50, 100 and 200 mg/kg of E. faecium respectively). The experiment lasted 42 days in two periods of 21 days. Results showed that there were no differences in breast meat quality among different treatments (p > 0.05). Compared with the CON and ANT treatments, the yellowness of thigh meat in E. faecium‐supplemented treatments was significantly increased (p < 0.05); the shear force of thigh meat in the LEF and MEF treatments was lower than that of the CON treatment (p < 0.05). In addition, the concentration of the inosine monophosphate (IMP) in the breast and thigh meat of the MEF treatment was significantly higher than that of the other treatments (p < 0.05). At 21 days, the total antioxidant capability (T‐AOC) level and glutathione peroxidase (GSH‐Px) activity of breast meat and superoxide dismutase (SOD) in the thigh meat of the MEF treatment were greatly increased (p < 0.05). At 42 days of age, the catalase (CAT), GSH‐Px and T‐AOC activities in the breast meat of the MEF treatment were increased (p < 0.05) and the CAT activity of thigh meat in the LEF and MEF treatments was increased (p < 0.05). In conclusion, E. faecium supplementation increased the meat quality of the thigh muscle, increased the IMP content and the activities of CAT, SOD, T‐AOC, and GSH‐Px of muscle in broilers. Supplementation with 100 mg/kg E. faecium had the greatest effects.     

屎肠球菌对AA肉鸡血清和肝脏抗氧化功能的影响

试验旨在研究日粮中添加屎肠球菌对爱拔益加（Arbor Acres,AA）肉鸡血清和肝脏抗氧化功能的影响。选取1日龄AA肉鸡600只（公鸡）,随机分为5组,每组6个重复。对照组（CON组）饲喂基础日粮;抗生素组（Ant组）在基础日粮中添加0.1%金霉素;低（LEF组）、中（MEF组）、高（HEF组）剂量屎肠球菌组分别在基础日粮中添加50、100、200 mg/kg屎肠球菌。结果表明：①21日龄时,HEF组肉鸡血清中总抗氧化能力（T-AOC）、谷胱甘肽过氧化物酶（GSH-Px）活性均显著高于CON组（P<0.05）,Ant组T-AOC显著高于CON组（P<0.05）;42日龄时,MEF组和HEF组血清中丙二醛（MDA）含量显著低于CON组（P<0.05）,LEF组血清中超氧化物歧化酶（SOD）活性显著高于CON组和Ant组（P<0.05）。②21日龄时,MEF组和HEF组肉鸡肝脏中T-AOC、GSH-Px活性均显著高于CON组（P<0.05）,且HEF组肉鸡肝脏中T-AOC显著高于Ant组（P<0.05）;42日龄时,HEF组肉鸡肝脏中过氧化氢酶（CAT）活性显著高于CON组（P<0.05）。结果提示,屎肠球菌能显著提高21与42日龄血清和肝脏中T-AOC和GSH-Px活性,降低血清中MDA含量,增加抗氧化酶活性,改善肉鸡抗氧化应激能力,且200 mg/kg屎肠球菌添加量在提高血清和肝脏抗氧化能力上与抗生素效果相当。     

粪产碱菌耐铵工程菌与水稻联合共生固氮作用

粪产碱菌（ Ａａｅｃａｌｉｓｆａｅｃａｌｉｓ） 在ＬＷ 培养基下能产生植物激素ＩＡＡ。盆栽结果表明,接种Ａ．ｆａｅｃａｌｉｓ 显著提高了水稻的生长,接种Ａ１５０１ 和Ａ１５１３ 的水稻稻谷产量分别比未接种提高了８５％ 和１０３ ％ ,用１５ Ｎ 同位素稀释法估测水稻地上部总氮中来源联合固氮的百分率达９００ ％ 和１１５ ％ ,其结果与水稻地上部（ 茎叶及稻谷） 总氮量的增加幅度（８６ ％ 和１１６ ％ ）基本一致。研究还表明Ａ１５１３ 耐铵工程菌的产ＩＡＡ作用以及对水稻的生长和产量、联合固氮作用均优于原始菌株Ａ１５０１ 。     

2022年北京市宠物源细菌耐药性监测浅析

为了解北京市宠物源细菌的抗菌药物耐药情况,2022年,笔者对北京市四个城区的四家宠物医院的犬、猫共计50份样本的肛拭子进行了研究。试验对样品中的大肠杆菌和肠球菌首先进行了分离培养和质谱鉴定,然后采用微量肉汤稀释法分析分离菌株的耐药表型。结果共分离出大肠杆菌25株、肠球菌25株（屎肠球菌14株、粪肠球菌11株）。大肠杆菌耐药率最高的2种抗菌药为四环素和氨苄西林,多重耐药菌占44%;肠球菌耐药情况较严重,粪肠球菌耐药率最高的抗菌药物为磺胺异噁唑,屎肠球菌耐药率最高的抗菌药物为磺胺异噁唑、头孢西丁和红霉素,二者多重耐药菌占分离株总数的100%。综上,北京地区宠物源大肠杆菌、肠球菌的耐药情况较为严峻,且多重耐药现象突出,需要加强对宠物抗菌药使用的监督与管理。