Engineering Disease Resistant Cattle

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk’s nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K

AIMS: To understand the potential use of bacteriophage K to treat bovine Staphylococcus aureus mastitis, we studied the role of whey proteins in the inhibition of the phage-pathogen interaction in vitro. METHODS AND RESULTS: The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in raw bovine whey and serum. Incubation of S. aureus with phage in whey showed that the bacteria are more resistant to phage lysis when grown in whey and also bovine serum. Whey collected from 23 animals showed a wide variation in the level of phage-binding inhibition. The role of the protein component of milk whey in this inhibition was established; treatment of the whey by heat, proteases and ultrafiltration removed the inhibitory activity. Brief exposure of S. aureus cells to whey, followed by resuspension in broth, also reduced phage binding. Microscopy showed the adhesion of extracellular material to the S. aureus cell surface following exposure to whey. Chromatographic fractionation of the whey demonstrated that the inhibitory proteins were present in the high molecular weight fraction. CONCLUSIONS: The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phage attachment and thereby hindered lysis. The inhibitory whey proteins are of high molecular weight in their native form and may sterically block phage attachment sites on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have implications for any future use of phage therapy in the treatment of mastitis, and other diseases, caused by S. aureus. This pathogen is predicted to be much more resistant to phage treatment in vivo than would be expected from in vitro broth culture experiments.     

噬菌体治疗肺炎克雷伯菌感染的研究进展

肺炎克雷伯菌是肠杆菌科家族中的一员,在各种环境中广泛存在,可导致诸如奶牛乳房炎在内的多种动物疫病,引起人类的肺炎、尿路感染、菌血症、伤口性感染和化脓性脓肿在内的多种临床感染。该菌对抗生素的耐受日趋严重,而且高毒力菌株不断出现,给该菌的防控带来了巨大挑战。噬菌体是一种裂解细菌的病毒,因其具有治疗耐药细菌感染的潜力而备受关注,世界各地均有使用噬菌体成功治疗耐药细菌感染的案例。本文基于国内外对肺炎克雷伯菌及其噬菌体的研究数据,综述了肺炎克雷伯菌的流行病学调查情况和噬菌体在治疗肺炎克雷伯菌感染方面的应用,以期为基于肺炎克雷伯菌噬菌体的抗菌研究和临床应用提供参考。     

Structural changes induced by a lytic bacteriophage make ciprofloxacin effective against older biofilm of Klebsiella pneumoniae

Bacteria have evolved multiple mechanisms, such as biofilm formation, to thwart antibiotic action. Yet antibiotics remain the drug of choice against clinical infections. It has been documented that young biofilm of Klebsiella pneumoniae could be eradicated significantly by ciprofloxacin treatment alone. Since age of biofilm is a decisive factor in determining the outcome of antibiotic treatment, in the present study biofilm of K. pneumoniae, grown for extended periods was treated with ciprofloxacin and/or depolymerase producing lytic bacteriophage (KPO1K2). The reduction in bacterial numbers of older biofilm was greater after application of the two agents in combination as ciprofloxacin alone could not reduce bacterial biomass significantly in older biofilms (P > 0.05). Confocal microscopy suggested the induction of structural changes in the biofilm matrix and a decrease in micro-colony size after KPO1K2 treatment. The role of phage associated depolymerase was emphasized by the insignificant eradication of biofilm by a non-depolymerase producing bacteriophage that, however, eradicated the biofilm when applied concomitantly with purified depolymerase. These findings demonstrate that a lytic bacteriophage alone can eradicate older biofilms significantly and its action is primarily depolymerase mediated. However, application of phage and antibiotic in combination resulted in slightly increased biofilm eradication confirming the speculation that antibiotic efficacy can be augmented by bacteriophage.     

Bacteriophage-mediated transduction of antibiotic resistance in enterococci

Aims: Temperate bacteriophages are bacterial viruses that transfer genetic information between bacteria. This phenomenon is known as transduction, and it is important in acquisition of bacterial virulence genes and antimicrobial resistance determinants. The aim of this study was to demonstrate the role of bacteriophages in gene transfer (antibiotic resistance) in enterococci. Methods and Results: Three bacteriophages from environmental samples isolated on pig host strains of Enterococcus gallinarum and Enterococcus faecalis were evaluated in transduction experiments. Antibiotic resistance was transferred from Ent. gallinarum to Ent. faecalis (tetracycline resistance) and from Ent. faecalis to Enterococcus faecium, Enterococcus hirae/durans and Enterococcus casseliflavus (gentamicin resistance). Conclusions: Bacteriophages play a role in transfer of antibiotic resistance determinants in enterococci. Significance and Impact of the Study: This study confirms previous suggestions on transduction in enterococci, in particular on interspecies transduction. Interspecies transduction is significant because it widens the range of recipients involved in antimicrobial resistance transfer.     

Bacteriophage and their lysins for elimination of infectious bacteria

When phages were originally identified, the possibility of using them as antibacterial agents against pathogens was immediately recognized and put into practise based on the knowledge available at the time. However, with the advent of antibiotics a decline in the use of phage as therapeutics followed. Phages did, however, become more useful in the study of fundamental aspects of molecular biology and in the diagnostic laboratory for the identification of pathogenic bacteria. More recently, the original application of phage as therapeutics to treat human and animal infections has been rekindled, particularly in an era where antibiotic resistance has become so problematic/commonplace. Phage lysins have also been studied and utilized in their own right as potential therapeutics for the treatment of bacterial infections. Indeed the past decade has seen a considerable amount of research worldwide focused on the engineering of phages as antibacterial agents in a wide range of applications. Furthermore, the US Food and Drug Administration and/or the US Department of Agriculture have recently approved commercial phage preparations to prevent bacterial contamination of livestock, food crops, meat and other foods. Such developments have prompted this review into the status of phage research as it pertains to the control of infectious bacteria.     

Isolation and characterization of two anti-staphylococcal bacteriophages specific for pathogenic Staphylococcus aureus associated with bovine infections

AIMS: The aim of this study was to isolate and characterize bacteriophages against bovine Staphylococcus aureus associated with mastitis. METHODS AND RESULTS: We describe the isolation of two anti-staphylococcal phages namely DW2 and CS1 from farmyard slurry. Both phages were characterized by electron microscopy and restriction analysis and shown to belong to the Siphoviridae family. CS1 and DW2 were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. These phages were compared with the previously characterized Myoviridae phage K. Infusion of a cocktail of all three phages at 10(8) PFU ml(-1) into live cow teats resulted in no detectable increase in somatic cell counts in milks indicating that the phages did not irritate the animal. CONCLUSION: Two new anti-staphylococcal phages CS1 and DW2 were isolated and characterized and tested for immunogenicity in animal teats. SIGNIFICANCE AND IMPACT OF THE STUDY: The phages isolated in this study are active against pathogenic S. aureus and may be incorporated into teat-dips or teat-washes as a non-antibiotic prophylaxis against staphylococcal bovine mastitis.     

青枯菌特异性噬菌体的研究进展与应用

烟草等茄科植物青枯病的防治是一个世界性难题,传统的化学防治、合理轮作、抗病品种等措施无法有效控制该病的发生。噬菌体用于细菌性病害的防治已有很长历史,近年来利用噬菌体防治青枯菌引发的青枯病方面的研究越来越受重视。我们简要综述了青枯菌噬菌体的研究进展,并对青枯菌噬菌体生物防治的应用前景进行了展望。     

Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk

AIMS: To assess the ability of staphylococcal bacteriophage K to inhibit Staphylococcus aureus in raw milk. METHODS AND RESULTS: The ability of bacteriophage (phage) to replicate in milk is important in situations where phage might be used as a therapeutic for bovine mastitis. Phage K was able to replicate normally, leading to elimination of the host culture in milk, which had been previously heat-treated. When raw milk was used under identical conditions, the phages were unable to replicate. Phage adsorption assays were performed and these demonstrated that adsorption of phage was significantly reduced in the raw milk while it was restored in the heat-treated sample (86.50% compared with 99.96% adsorption respectively). When confocal microscopy with a Live/Dead Bac light staining system was employed, it was observed that in raw milk S. aureus formed clusters associated with fat globules, while in heat-treated milk, bacterial agglutination had not occurred. CONCLUSIONS: Raw milk inhibits staphylococcal phage K proliferation. Significance and Impact of the Study: This observation has implications for the exploitation of staphylococcal therapeutic phage in milk.     

一株可裂解动物源性多重耐药志贺氏菌的噬菌体分离鉴定及其生物学特性研究

从鸡的肠道内分离鉴定多重耐药志贺氏菌(Shigelle),纯化获得可侵袭、裂解该志贺氏菌的噬菌体,并研究其生物学特性。以养殖场病鸡肠道中分离的志贺氏病原菌为宿主,筛选鉴定噬菌体,聚乙二醇沉淀浓缩噬菌体,通过透射电镜观察噬菌体形态,双层平板法测定噬菌体宿主谱、最佳感染复数、一步生长曲线、酸碱耐受性和热稳定性,测定噬菌体对多重耐药致病性志贺氏菌的裂解效果。通过上述方法,共筛选到26株志贺氏菌分离株,分别命名为BS1~BS26。以志贺氏菌BS26为宿主菌分离得到一株裂解性噬菌体并命名为噬菌体PSF26,鉴定为有尾噬菌体目,长尾噬菌体科,噬菌体对福氏志贺氏菌（Shigella flexneri）和宋内志贺氏菌(Shigella sonnei)分离株均有裂解作用。噬菌体PSF26的最佳感染复数为0.1,感染周期为90 min,包括75 min的潜伏期和15 min的爆发期,裂解量为(58±17) pfu/cell。噬菌体PSF26在pH 5~8,温度0~40 ℃条件下能保持较高活性,对宿主菌的抑菌圈直径为(11.33±1.53) mm。结果表明,噬菌体PSF26爆发期短,裂解量高,有较好的酸碱、温度耐受性,对多重耐药性福氏和宋内志贺氏菌有良好的作用效果,为利用噬菌体防控饲养动物中食源性致病菌感染和饲用抗生素替代技术研发提供参考。     

我国奶牛乳房炎致病性金黄色葡萄球菌血清型分布

本试验采用玻片凝集法对从临床型乳房炎病乳中分离获得的56株金黄色葡萄球菌进行血清型分型。结果表明336型占67.9%(38/56),5型占7.1%(4/56),8型占3.6%(2/56)。