Genome analysis of methicillin‐resistant Staphylococcus aureus isolated from pigs: Detection of the clonal lineage ST398 in Cameroon and South Africa

Food animals are considered reservoirs of methicillin‐resistant Staphylococcus aureus (MRSA) and are implicated in their zoonotic transmission in the farm‐to‐plate continuum. LA‐MRSA has been reported as a zoonotic agent that has the potential to spread to humans and may cause infections in at‐risk groups. In this study, whole genome sequencing was used to describe the genetic environment (resistance mechanisms, virulence factors and mobile genetic elements) and investigate the genetic lineages of MRSA isolates from pigs in Cameroonian and South African abattoirs. During March–October 2016, 288 nasal and rectal pooled samples from 432 pigs as well as nasal and hand swabs from 82 humans were collected. Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were de novo‐assembled using the Qiagen CLC Genomics Workbench and SPAdes. The assembled contigs were annotated, and antibiotic resistance genes, virulence factors, plasmids, SCCmec and phage elements were identified with ResFinder, Virulence Finder, PlasmidFinder, SCCmec Finder and PHAST, respectively. Core genome single nucleotide analysis was undertaken to assess clonal relatedness among isolates. A lower MRSA prevalence was observed in pigs in Cameroon (n = 1/13; 0.07%) compared with South Africa (n = 4/22; 18.18%), and none of the workers were colonized by MRSA. Genome analysis identified various antibiotic resistance genes along with six virulence factors in all isolates. All MRSA isolates belonged to the clonal lineage ST398 (spa‐type t011) and harboured the type Vc SCCmec and several plasmids. Our study shows that the livestock‐associated MRSA clonal lineage ST398 is already present in both Cameroon and South Africa and is probably underestimated in the absence of molecular epidemiological studies. It reveals the serious food safety and public health threat associated with this animal strain and underscores the need for interventions to contain this resistant clone.     

抗菌肽NZ2114对奶牛乳房炎源金黄色葡萄球菌细胞膜、基因组和耐药性的影响

本试验以真菌防御素Plectasin衍生肽NZ2114和奶牛乳房炎源金黄色葡萄球菌为研究对象,旨在阐明抗菌肽NZ2114对金黄色葡萄球菌的体外抗菌机制及其阻遏金黄色葡萄球菌耐药性的效果。通过16S rRNA基因鉴定对55份乳房炎奶样中的病原菌进行分离鉴定,采用纸片扩散法检测其耐药性,通过微量稀释法测定抗菌肽NZ2114对金黄色葡萄球菌的抑菌活性,借助流式细胞术和扫描电镜观察抗菌肽NZ2114对金黄色葡萄球菌细胞膜完整性及细胞形态的影响,通过凝胶阻滞和圆二色谱分析抗菌肽NZ2114对金黄色葡萄球菌基因组DNA的影响,将抗菌肽NZ2114与金黄色葡萄球菌共同孵育以研究抗菌肽NZ2114对金黄色葡萄球菌耐药性的影响,通过PCR进一步分析抗菌肽NZ2114对金黄色葡萄球菌携带的耐药基因的影响。结果表明,共获得10株金黄色葡萄球菌,分离菌对青霉素等9种抗菌药具有耐药性,且50%为多重耐药。抗菌肽NZ2114对10株金黄色葡萄球菌具有强抑菌活性,最小抑菌浓度（MIC）为0.5~1.0 μg/mL。抗菌肽NZ2114可导致金黄色葡萄球菌S7细胞皱缩、细胞内容物泄漏,甚至细胞裂解,碘化丙啶（PI）细胞膜穿透率达5%,且抗菌肽NZ2114可与金黄色葡萄球菌S7基因组DNA结合并改变其DNA结构。金黄色葡萄球菌与1/4×MIC抗菌肽NZ2114孵育12 h后发现,除对阿莫西林和磺胺异噁唑的耐药率没有降低外,对其他抗菌药的耐药率均有不同程度的降低（10%~40%）,且抗菌肽NZ2114对β-内酰胺类耐药基因（blaZ）及杆菌肽类耐药基因（braRS）的消除率分别达28.57%（2/7）和22.22%（2/9）。上述结果证明,抗菌肽NZ2114对耐药金黄色葡萄球菌具有体外强抑菌活性,其干扰耐药金黄色葡萄球菌细胞膜并结合胞内DNA的作用机制为其低耐药杀菌机制奠定了细胞学基础。同时抗菌肽NZ2114对耐药菌株的耐药性及相关耐药基因均有一定消除作用。由此可见,抗菌肽NZ2114是一种极具前景的治疗金黄色葡萄球菌引起的奶牛乳房炎的抗生素替代品。     

奶牛乳房炎病原菌金黄色葡萄球菌的分离鉴定及耐药性分析

为研究江苏某奶牛场金黄色葡萄球菌的耐药特点,本试验通过MALDIBiotyper系统对8株金黄色葡萄球菌疑似株进行鉴定和耐药性试验。结果显示,8株金黄色葡萄球菌疑似株均为金黄色葡萄球菌,其对青霉素、链霉素、环丙沙星的耐药率均为100%;其次,对阿莫西林的耐药率为75%;极少数对头孢氨苄、庆大霉素、丁胺卡那、卡那霉素耐药;而对四环素、多西环素、克林霉素均敏感。结果表明,该奶牛场金黄色葡萄球菌的分离率为14.55%(8/55),且均呈现多重耐药。     

驴源短小芽孢杆菌LV2分离鉴定及其生物学特性

通过分离培养和形态特征从3头12月龄健康德州驴盲肠内容物样品中分离到芽孢杆菌,采用生理生化和分子生物学方法鉴定分离菌,研究了分离菌的生长曲线、产纤维素酶活性、抗菌活性、药物敏感性及安全性等生物学特性。结果显示,分离到革兰阳性芽孢杆菌1株,鉴定为短小芽孢杆菌,编号为LV2;该菌可降解羧甲基纤维素,产生清晰透明圈,对奶牛乳房炎金黄色葡萄球菌具有较强的抑制作用;在测定的20种临床常用抗生素中仅对林可霉素表现为中度敏感,对其他抗生素均表现为敏感;急性毒性试验和慢性毒性试验结果表明该菌株安全性良好。结果表明,本试验分离到的驴源短小芽孢杆菌具有安全、产纤维素酶、抑制金黄色葡萄球菌等益生活性,在研发驴用微生态制剂或生物发酵粗饲料领域具有潜在的开发价值。     

牛源金黄色葡萄球菌凝固酶分型与致病因子检测

为了解青海和甘肃地区奶牛养殖场的金黄色葡萄球菌(S.aureus)基因型分布状况,本研究对来自该两个地区的210份奶样进行了S.aureus的分离与鉴定,利用凝固酶基因(Coa)扩增和限制性内切酶AluⅠ酶切方法对所有分离株进行了凝固酶多态性分型。并对不同凝固酶基因型的S.aureus进行了白细胞毒素F (LukF)、白细胞毒素S (LukS)、α溶血素(α-hemolysin)、β溶血素(β-hemolysin)等主要致病因子检测。结果显示,共分离鉴定出35株S.aureus (青海13株,甘肃22株),所有分离株均能够扩增出Coa基因片段,并有5种PCR分型结果(本文简称PCR1～PCR5型),其中PCR 1、4和5型只有1种亚型,而PCR 2型有3种亚型,PCR 3型有2种亚型。PCR3型是青海(6/13)和甘肃地区(8/22)主要流行的基因型。致病因子检测结果显示,所有分离株均携带致病因子LukS和α溶血素;致病因子LukF和β溶血素检出率也很高,分别是30株(85.7%)和31株(88.6%)。β溶血素基因在甘肃地区检出率为95.5%(21/22),青海地区检出率为76.9%(10/13),致病因子LukF在甘肃地区检出率为91.0%(20/22),青海地区检出率为76.9%(10/13),且在各基因型中均有分布,甘肃地区S.aureus致病因子LukF和β溶血素检出率高于青海地区。本研究首次对青海和甘肃地区致奶牛乳房炎S.aureus进行了鉴定分析,为这两个地区奶牛乳房炎的防治提供了参考依据。     

赤芍水提物对金黄色葡萄球菌肠毒素及蛋白A表达的影响

探讨了赤芍水提物在体外对金黄色葡萄球菌肠毒素A、B及蛋白A分泌及mRNA转录的影响。试验中采用微量倍比稀释法测定赤芍对金黄色葡萄球菌ATCC29213的最小抑菌浓度(MIC);采用Western blot和实时荧光定量PCR检测亚抑菌浓度下赤芍水提物对金黄色葡萄球菌肠毒素A、B及蛋白A在mRNA转录和毒素蛋白分泌表达的影响。结果表明,赤芍水提物对金黄色葡萄球菌ATCC29213的MIC值为6.3mg/mL;在亚抑菌浓度下,赤芍水提物可以明显抑制肠毒素A、B及蛋白A在蛋白水平和转录水平的表达,且呈现明显的浓度依赖性。     

武夷岩茶茶多酚抑菌作用的研究

用纸片法测定了1.0、2.5、5.0、10mg/mL 4种浓度的武夷岩茶茶多酚对大肠杆菌、致病性大肠杆菌、李斯特氏沙门氏菌、金黄色葡萄球菌等4种菌的抑菌效果。实验结果表明,茶多酚对4种菌均有明显的抑菌作用,其中对金黄色葡萄球菌的抑菌作用最强（P〈0.01）,4种浓度的抑菌圈分别为1.82±0.44～3.34±0.18（cm）;对李斯特氏沙门氏菌、大肠杆菌、致病性大肠杆菌的抑菌作用次之,抑菌圈分别为1.73±0.18～2.05±0.14（cm）;1.46±0.33～1.85±0.32（cm）;1.1±0.14～1.61±0.25（cm）。1mg/mL的茶多酚对4种菌的抑菌作用均小于同剂量的青霉素和庆大霉素（P〈0.01）。     

如何进行奶牛乳房炎金黄色葡萄球菌疫苗的合理评估

奶牛乳房炎是造成全球奶业经济损失的主要原因,而金黄色葡萄球菌是最主要的病原菌。本文系统、直观地对研究奶牛乳房炎金黄色葡萄球菌疫苗的科研结论进行量化总结和评估,目的是为奶牛乳房炎疫苗的研发提供新思路,为设计合理的奶牛乳房炎疫苗给动物评估试验方案提供科学依据。本文在Pubmed、Science数据库和CNKI数据库对＂奶牛乳房炎疫苗＂类的文章进行了合理的电子检索,然后对细菌疫苗、细菌类毒素疫苗、DNA-重组蛋白疫苗、单一的重组蛋白疫苗等方面的科研论文从试验设计、方法、疫苗类型和研究结果 4个方面进行了全面的分析。结果表明,采用DNA、重组蛋白新技术的疫苗和传统的细菌疫苗已取得良好效果,疫苗可以成为预防和控制金黄色葡萄球菌性奶牛乳房炎的最好或最有前景的一种途径。但是研究方法差异和双盲试验的缺乏阻碍对疫苗效果的合理评估。     

A case of a periarticular abscess and suppurative arthritis of the atlanto-occipital joint

This Case Report describes a periarticular abscess and a suppurative arthritis of the atlanto-occipital joint in an adult horse. The horse showed a painful swelling localised on the atlanto-occipital region and ataxia in all 4 limbs. During hospitalisation, the horse developed recumbency, dysphagia, facial paralysis and seizures. Ultrasonography of the atlanto-occipital region enabled a tentative diagnosis to be made, which was later confirmed at autopsy. Culture of the abscess revealed a Staphylococcus aureus. However, the horse had a history of recurrent fever, hypertrophy and abscessation of the submandibular lymph nodes, neck pain and dyspnoea for 2 months, which was suggestive of strangles.     

金黄色葡萄球菌IsdB基因缺失序列比较与宿主特异性分析

对新疆地区分离的牛源金黄色葡萄球菌XJ43(CC97)、XJ108(CC398)和XJ69(CC479)的IsdB基因序列测定、比对发现,在编码非功能结构域的277-314碱基(5’-3’)间存在不同序列的缺失,并导致各编码蛋白质的二级结构出现一定差异;Blast分析显示,NCBI中登录的金黄色葡萄球菌不同菌株IsdB基因序列相似性大于95%,但与新分离的三种序列型相比,除医源菌株USA300 TCH959(CC8)与XJ69(CC479)的IsdB基因序列缺失一致外,其他医源菌株与XJ43缺失的序列均不一致;牛源或羊源CC133、CC425、CC151和属人畜共患CC398 XJ108菌株IsdB基因缺失的序列一致,表明这种序列缺失与金黄色葡萄球菌的宿主特异性无绝对相关性,但含有IsdB基因序列缺失的菌株在反刍动物乳腺感染中出现频率较高。