金黄色葡萄球菌处理对奶牛乳腺上皮细胞外泌体表征的影响

【目的】本研究旨在从金黄色葡萄球菌(Staphylococcus aureus,S.aureus,以下简称金葡菌)处理奶牛乳腺上皮细胞(bovine mammary epithelial cell,BMEC)上清中分离外泌体,探究金葡菌与BMEC数量比值(MOI)和处理后培养时间对外泌体总浓度的影响,并对金葡菌诱导BMEC释放的外泌体进行分离和鉴定。【方法】采用金葡菌临床分离菌株以MOI＝1和MOI＝10分别处理BMEC 3 h,对已处理的BMEC继续无外泌体培养9、12、24 h,同时设空白对照组,通过扫描电镜观察金葡菌处理对BMEC造成的超微结构损伤。采用超速离心法分离金葡菌诱导的BMEC上清液中的外泌体,分别用透射电镜、纳米颗粒追踪分析技术及Western blotting法对外泌体形态、颗粒大小和特异性标志蛋白进行分析鉴定。通过检测外泌体总蛋白浓度评估不同MOI和处理后培养时间对外泌体总量的影响,明确金葡菌诱导BMEC释放外泌体的最佳条件。【结果】通过扫描电镜观察发现,金葡菌处理BMEC后,细胞微绒毛脱落,细胞骨架破坏。透射电镜观察分离的外泌体为圆形的双层膜囊泡,直径在30~150 nm,形态均一。通过检测外泌体总蛋白浓度显示,在MOI＝10时,培养9、12和24 h金葡菌诱导BMEC释放外泌体蛋白浓度均高于MOI＝1,而培养12 h时释放外泌体蛋白浓度最高。对金葡菌诱导BMEC释放的外泌体进一步鉴定,其平均直径约为116 nm;Western blotting检测结果显示外泌体标记物CD9、CD81和TSG101表达阳性。【结论】本研究从形态学和分子生物学特征等方面证实获得的分离物为外泌体;金葡菌可以诱导BMEC释放外泌体,当MOI＝10时,金葡菌处理细胞3 h后继续无外泌体培养12 h,收获的细胞上清中外泌体蛋白含量最高。     

Culture results from milk samples submitted to veterinary diagnostic laboratories from August 2003 to December 2006 in New Zealand

Abstract

AIMS: To determine the pattern of isolation of major mastitis-causing organisms isolated from milk samples submitted to five veterinary diagnostic laboratories in New Zealand.

METHODS: The culture results of 25,288 milk samples that were collected from dairy cows throughout New Zealand from August 2003 to December 2006 and submitted to a group of veterinary diagnostic laboratories were assembled, reviewed and summarised. Logistic regression was used to analyse the effect of year, region (i.e. North vs South Island), and season on the probability of isolating the two most common organisms.

RESULTS: The most commonly isolated mastitis causing organisms from all samples were: Streptococcus uberis (23.6%), Staphylococcus aureus (23.5%), coagulase-negative staphylococci (CNS; 7.2%), Strep. dysgalactiae (6.2%), Bacillus spp. (4.0%), and coliforms (3.7%). The percentage of samples with isolates of Strep. uberis or Staph. aureus was affected by island, year and season (p<0.001). For most of the year, except in late winter and early spring when Strep. uberis was much more common, the percentage of isolates of Strep. uberis and Staph. aureus were not apparently different despite the former being an environmental pathogen and the other a contagious one.

CONCLUSION: The pattern of isolation of major mastitis-causing organisms, as determined from culture of milk samples submitted to diagnostic laboratories in New Zealand, has changed significantly over the last 40 years, with a substantial increase in the percentage of isolates that are Strep. uberis and a decrease in isolates of Strep. agalactiae. There is a clear seasonal pattern to the isolation of both Strep. uberis and Staph. aureus, particularly the former.

CLINICAL RELEVANCE: Knowledge of the aetiological agents causing bovine mastitis on a farm is of value in determining the choice of treatment. This dataset shows that, although there is seasonal pattern to the isolation of mastitis-causing organisms in New Zealand, both Strep. uberis and Staph. aureus are isolated throughout the year, so bacteriology is of value in determining aetiology even in late winter/early spring.     

Prevalence and antimicrobial resistance of MRSA across different pig age groups in an intensive pig production system in Australia

This observational study aimed to determine MRSA prevalence using strain‐specific real‐time PCR at the pig level, stratified by age groupings, within a pig enterprise. A total of 658 samples were collected from individual pigs (n = 618) and the piggery environment (n = 40), distributed amongst five different pig age groups. Presumptive MRSA isolates were confirmed by the presence of mecA, and MALDI‐TOF was performed for species verification. All isolates were tested against 18 different antimicrobials. MRSA was isolated from 75.2% (95% CI 71.8–78.6) of samples collected from pigs, and 71% of the MRSA isolates from this source were identified as community‐associated (CA)‐MRSA ST93, while the remainder were livestock‐associated (LA)‐MRSA ST398. Amongst environmental isolates, 80% (CI 64.3–95.7) were ST93 and the remainder ST398. All MRSA isolates from pigs and the environment were susceptible to ciprofloxacin, gentamicin, linezolid, mupirocin, rifampicin, sulfamethoxazole–trimethoprim, teicoplanin and vancomycin. Phenotypic rates of resistance were penicillin (100%), clindamycin (97.6%), erythromycin (96.3%), ceftiofur (93.7%), chloramphenicol (81.2%), tetracycline (63.1%) and amoxicillin–clavulanate (63.9%). A low prevalence of resistance (9.2%) was observed against neomycin and quinupristin–dalfopristin. The probability of MRSA carriage in dry sows (42.2%) was found to be significantly lower (p < .001) when compared to other age groups: farrowing sows (76.8%, RR1.82), weaners (97.8%, RR 2.32), growers (94.2%, RR 2.23) and finishers (98.3%, RR 2.33). Amongst different production age groups, a significant difference was also found in antimicrobial resistance for amoxicillin–clavulanate, neomycin, chloramphenicol and tetracycline. Using the RT‐PCR assay adopted in this study, filtering of highly prevalent ST93 and non‐ST93 isolates was performed at high throughput and low cost. In conclusion, this study found that weaner pigs presented a higher risk for CA‐MRSA and antimicrobial resistance compared to other age groups. These findings have major implications for how investigations of MRSA outbreaks should be approached under the One‐Health context.     

固体脂质纳米增强抗菌药物对胞内金黄色葡萄球菌活性的研究进展

金黄色葡萄球菌导致的乳房炎是危害奶牛养殖业最严重的疾病之一。抗菌药在胞内极易排出或失活,导致奶牛金黄色葡萄球菌乳房炎治疗效果差且容易复发。纳米粒子具有在巨噬细胞和上皮细胞等多种细胞中积累的能力,可提高药物在胞内的浓度和胞内停留时间,这使得它们可用于治疗细胞内感染。本文对固体脂质纳米抗金黄色葡萄球菌感染的研究进展进行综述,以指导和推动固体脂质纳米在治疗金黄色葡萄球菌乳腺炎方面的应用。     

奶牛乳房炎金黄色葡萄球菌疫苗研究进展

奶牛乳房炎是影响乳品工业经济效益的重要疾病之一,引起奶牛乳房炎的致病菌约有150多种,但金黄色葡萄球菌是引起奶牛乳房炎的主要病原。由它引起的乳房炎很难治愈,而且对抗生素产生耐药性的菌株已经相当普遍,因此使用预防或治疗性疫苗防治乳房炎就成为首选。然而金黄色葡萄球菌的致病力由许多不同的因子(如黏附素、荚膜等)决定,这使得制备非常有效的疫苗变得很困难。本文就这些致病因子在疫苗研制方面的进展做一简要介绍。     

金黄色葡萄球菌对奶牛乳腺上皮细胞Wnt/β-catenin信号通路和相关修复因子的影响

采用金黄色葡萄球菌侵袭原代奶牛乳腺上皮细胞,观察该病原菌对乳腺上皮细胞Wnt/β-catenin信号通路关键蛋白和相关修复因子EGFR、TGF-β3和VEGF基因mRNA表达的影响。金黄色葡萄球菌以MOI=1∶1的比例接种奶牛乳腺上皮细胞,分别作用0,15,30,45,60,120,240 min,采用Western blot法检测乳腺上皮细胞β-catenin、Cyclin D1及c-Myc蛋白表达水平;免疫荧光法检测β-catenin的表达及核易位;荧光定量PCR检测EGFR、TGF-β3和VEGF基因mRNA表达。结果显示,β-catenin蛋白在45,60,120 min表达量升高,与0 min相比差异显著(P<0.05);Cyclin D1蛋白在45,60,120和240 min表达量升高,差异显著或极显著(P<0.05或P<0.01);c-Myc蛋白在15,30,60,120,240 min表达量升高,差异极显著(P<0.01)。修复相关因子EGFR、TGF-β3和VEGF基因mRNA的表达量在30 min均出现极显著升高(P<0.01),且VEGF基因mRNA在45,60,120和240 min均呈现极显著升高(P<0.01),EGFR基因mRNA表达量在30,45和60 min时表达量极显著升高(P<0.01)。结果表明,金黄色葡萄球菌侵袭奶牛乳腺上皮细胞后,诱导Wnt/β-catenin信号通路的转导和修复因子EGFR、TGF-β3和VEGF基因转录,该信号通路和修复因子可能参与金黄色葡萄球菌导致的炎症和细胞损伤的修复过程。     

Methicillin‐resistant Staphylococcus aureus in urban Norway rat (Rattus norvegicus) populations: Epidemiology and the impacts of kill‐trapping

Urban Norway rat (Rattus norvegicus) populations can carry the bacteria methicillin‐resistant Staphylococcus aureus (MRSA). There are numerous knowledge gaps in the epidemiology of MRSA in these populations that limit understanding of its ecology in urban environments. For example, fecal shedding of MRSA, which may increase environmental contamination, has been reported in other species; however, it is unknown whether Norway rats carry the bacteria rectally. Furthermore, while intermittent MRSA shedding has been shown in other species and may dictate when the risk of transmission is highest, duration of carriage has not been examined for Norway rats. Previous work has shown that lethal animal‐control methods may increase the level of pathogens within reservoir populations, possibly by disrupting ecological patterns. However, the impact of rodent‐control on potentially environmentally acquired pathogens like MRSA has not been tested. Using capture‐mark‐recapture methods in an inner‐city neighborhood in Vancouver, Canada, we show that rats intermittently carry MRSA both in the rectum and oropharynx. By assessing the prevalence of MRSA before and after enacting a pest‐control intervention, we report that kill‐trapping had no impact on the prevalence of carriage of this environmentally‐acquired agent.     

实验动物粪便中金黄色葡萄球菌和沙门菌检验方法的比较

为探讨实验动物粪便中金黄色葡萄球菌和沙门菌的检测方法,试验比较了国标的方法、ATB生化鉴定试纸条、rRNA测序分析对样品的检测结果,发现生化鉴定试纸条和16S rRNA测序分析均可以准确、高效地检测出沙门菌和金黄色葡萄球菌,而参考国标的方法,有些金黄色葡萄球菌和沙门菌的鉴定结果与国标不一致。试验表明,用ATB生化鉴定试纸条和16S rRNA序列分析检测沙门菌和金黄色葡萄球菌及其他菌的方法更可靠。     

Field trial of a staphylococcal mastitis vaccine in dairy herds: clinical, subclinical and microbiological assessments

Objective To assess the efficacy of a new staphylococcal mastitis vaccine under commercial dairying conditions.
Design A field trial involving 1819 cows and heifers conducted on seven dairy herds in Victoria. The trial was done 'blind'; approximately half the animals were vaccinated and the remainder were untreated controls.
Procedure The vaccine was given twice during the last 10 weeks of pregnancy. Effects of vaccination were assessed, during the ensuing lactation, on the basis of clinical and sub-clinical mastitis and microbiological investigations of the milk.
Results A total of 273 cases of clinical mastitis were recorded. Staphylococcus aureus was isolated from 112 of these, 45 cases in vaccinates and 67 cases in controls; the difference was not statistically significant. One herd was notable in having a high incidence of clinical staphylococcal mastitis. This herd accounted for 15.8% of the animals in the field trial but 54.5% of cases of clinical staphylococcal mastitis. For this herd, vaccinated animals had significantly lower incidence of clinical staphylococcal mastitis and prevalence of subclinical mastitis, relative to controls. An unexpected feature of the trial as a whole was the low incidence of clinical mastitis from which S aureus was isolated in pure culture (26.3% of cases) and the high incidence of clinical Streptococcus uberis mastitis (22.7% of cases).
Conclusions The trial showed that the vaccine was efficacious in reducing the incidence of clinical mastitis and prevalence of subclinical mastitis in a herd that had a serious staphylococcal mastitis problem.     

Low prevalence of zoonotic multidrug‐resistant bacteria in veterinarians in a country with prudent use of antimicrobials in animals

The occurrence of multidrug‐resistant zoonotic bacteria in animals has been increasing worldwide. Working in close contact with livestock increases the risk of carriage of these bacteria. We investigated the occurrence of extended‐spectrum beta‐lactamase (ESBL) and plasmidic AmpC beta‐lactamase producing Enterobacteriaceae (ESBL/pAmpC‐PE) and livestock‐associated methicillin‐resistant Staphylococcus aureus (LA‐MRSA) in Finnish veterinarians (n = 320). In addition to microbiological samples, background information was collected. Bacterial whole genome sequencing was performed to deduce sequence types (STs), spa types and resistance genes of the isolates. In total, 3.0% (9/297) of the veterinarians carried ESBL producing Escherichia coli, with one ESBL producing E. coli isolate producing also AmpC. Seven different STs, sequences of several different plasmid groups as well as several different bla_ESBL/pAmpC genes existed in different combinations. No carbapenemase or colistin resistance genes were detected. MRSA was detected in 0.3% (1/320) of the samples. The strain belonged to LA‐MRSA clonal complex (CC) 398 (ST398, spa type 011, lacking Panton‐Valentine leukocidin genes). In conclusion, this study shows low carriage of multidrug‐resistant zoonotic bacteria in Finnish veterinarians. However, finding LA‐MRSA for the first time in a sample from a veterinarian in a country with prudent use of animal antimicrobials and regarding the recent rise of LA‐MRSA on Finnish pig farms, a strong recommendation to protect people working in close contact with animals carrying LA‐MRSA CC398 is given. Further studies are needed to explain why the prevalence of LA‐MRSA in veterinarians is lower in Finland than in other European countries.