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。     

Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome

Based on our clinical experience on bovine mastitis, we hypothesized that subtypes of Staphylococcus aureus (S. aureus) exist which differ in their contagious and pathogenic properties. In order to investigate this hypothesis, we analyzed strains of S. aureus isolated from spontaneous intramammary infection (IMI) with their virulence gene patterns and genotypes obtained by PCR amplification of the 16S-23S rRNA intergenic spacer (RS-PCR). The genotypes were then associated with epidemiological and clinical data including 26 herds. The results demonstrated a high association between genotypes and virulence gene patterns as well as between epidemiological and pathogenic properties of S. aureus. In particular, genotype B was related to high contagiosity and increased pathogenicity whereas the other types (C, OG) were found with infection of single cows. Because of the high clinical relevance, our results indicate the need to subtype the IMI-associated strains of S. aureus in the future.     

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。     

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.     

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。     

Subclinical mastitis changes the patterns of maternal-offspring behaviour in dairy sheep

Subclinical mastitis was induced by inoculating one mammary gland in dairy ewes (n=8) with a Staphylococcus simulans isolate; control ewes (n=4) were included. The milk yield of inoculated glands decreased (P<0.001), the California Mastitis Test (CMT) score increased and the organism could be recovered from the inoculated glands. With time, there was significantly increased frequency of "hindering sucking" (P=0.016) and "head up posture" (P<0.001) in the control ewes. Infected ewes had a significantly increased frequency of "vocalisation" (P=0.013) compared to controls. There was a significant difference in the frequency of "sucking attempt" and "successful suck" (P<0.05) behaviours between lambs of the two groups. Lambs of the challenged ewes also showed significantly increased frequency and duration of these behaviours towards the uninoculated glands of their dams, rather than to the challenged glands (P<0.05); no such difference was evident for the lambs of control ewes. It was concluded that subclinical mastitis alters the sucking behaviour of both ewes and lambs.     

Distribution of the putative virulence factor encoding gene sheta in Staphylococcus hyicus strains of various origins

In the present study, Staphylococcus (S.) hyicus strains isolated in Russia (n = 23) and Germany (n = 17) were investigated for the prevalence of the previously described genes sheta and shetb. Sheta was detected in 16 S. hyicus strains. Sheta-positive strains were mainly found among strains isolated from exudative epidermitis, and frequently together with the exfoliative toxin-encoding genes exhD and exhC. Partial sequencing of sheta in a single S. hyicus strain revealed an almost complete match with the sheta sequence obtained from GenBank. None of the S. hyicus strains displayed a positive reaction with the shetb-specific oligonucleotide primer used in the present study. According to the present results, the exotoxin encoding gene sheta seems to be distributed among S. hyicus strains in Russia and Germany. The toxigenic potential of this exotoxin, which does not have the classical structure of a staphylococcal exfoliative toxin, remains to be elucidated.     

PRELIMINARY EVALUATION OF COMPOSTED PISTACHIO HULL AND RICE HUSK AS POTTING MEDIUM ON SCINDAPSUS AUREUS VEGETATIVE AND PHYSIOLOGICAL CHARACTERISTICS IN GREENHOUSE CONDITION

Pistachio hull is a horticultural waste product obtained during dehulling process of pistachio nuts. This experiment was carried out to evaluate the effects of pistachio hull and rice husk compost, as culture medium, on vegetative and physiological characteristics of pothos (Scindapsus aureus) plants. Rooted cuttings were used as plant materials and cultured in pots (40 × 30cm) filled with the following media: S₁ = 100% peat (control), S₂ = 100% rice husk, S₃ = 70% rice husk + 30% pistachio hull, S₄ = 50% rice husk + 50% pistachio hull, S₅ = 30% rice husk + 70% pistachio hull and S₆ = 100% pistachio hull. The vegetative parameters were affected by media type, so that no plants survived in 100% pistachio hull. The application of 50% pistachio hull and 50% rice husk (S₄), increased shoot fresh weight, and potassium (K) concentration of shoot and root compared to control. Fresh and dry root weight and phosphorus (P) concentration of shoot was increased with S₂ treatment (100% rice husk).     

Interpretation of findings leptospirosis

Extract

Sir,—In his criticism of our recent paper in which we claimed to have demonstrated experimental leptospirosis in 19 of the 20 control calves, W. M. Webster claims that the evidence for infection in five of the control calves is unconvincing (“Standards in Scientific Contributions”, N.Z. vel. J., 6: 27). His main reasons for this opinion appear to be, first, because no significant agglutination-lysis titres were demonstrated in these calves and, secondly, the observed onset of leptospimria in three of them was later than nsnal. He further suggests that mistaken diagnosis of leptospirosis, due either to dark-ground examination of urine or to inadvertent infection in guinea pigs inoculated with calf kidney tissue, is a more probable explanation.     

Brain microabscesses in a porcine model of Staphylococcus aureus sepsis

Background

Sepsis caused by Staphylococcus aureus often leads to brain microabscesses in humans. Animal models of haematogenous brain abscesses would be useful to study this condition in detail. Recently, we developed a model of S. aureus sepsis in pigs and here we report that brain microabscesses develop in pigs with such induced S. aureus sepsis.Twelve pigs were divided into three groups. Nine pigs received an intravenous inoculation of S. aureus once at time 0 h (group 1) or twice at time 0 h and 12 h (groups 2 and 3). In each group the fourth pig served as control. The pigs were euthanized at time 12 h (Group 1), 24 h (Group 2) and 48 h (Group 3) after the first inoculation. The brains were collected and examined histopathologically.

Results

All inoculated pigs developed sepsis and seven out of nine pigs developed brain microabscesses. The microabscesses contained S. aureus and were located in the prosencephalon and mesencephalon. Chorioditis and meningitis occurred from 12 h after inoculation.

Conclusions

Pigs with experimental S. aureus sepsis often develop brain microabscesses. The porcine brain pathology mirrors the findings in human sepsis patients. We therefore suggest the pig as a useful animal model of the development of brain microabscesses caused by S. aureus sepsis.