Data Sheet 1_Controlling drug-resistant bacteria in Arabian horses: bacteriophage cocktails for treating wound infections.docx

Antimicrobial resistance is a major global health issue requiring a coordinated response. This study investigated for the first time the prevalence, antimicrobial resistance phenotypes of bacteria causing infections in Arabian horses, and the potential of bacteriophage therapy for wound treatment. One hundred clinical samples from infected Arabian horses, presenting respiratory disorders, diarrhea, abortion, wound, and ocular infection, were examined using direct sample multiplex PCR and phenotypic methods. Antimicrobial susceptibility testing of the recovered isolates was performed using panels of 37 antibiotics and broth microdilution method. Bacteriophages were isolated from horse manure. A bacteriophage cocktail was used for treating infected wounds in Arabian horses. Streptococcus equi was the most predominant pathogen isolated from respiratory infections (17/29, 58.6%), followed by Klebsiella pneumoniae and Pseudomonas aeruginosa (9/29, 31.03%, each), and Escherichia coli (7/29, 24.13%). Staphylococcus aureus and Corynebacterium ovis biovar equi were the most frequently isolated bacteria from pyogenic infections. All isolated bacteria showed resistance to multiple antibiotics. Streptococcus spp. exhibited extensive drug resistance (XDR) with complete resistance to amoxicillin-clavulanic acid, amikacin, kanamycin, streptomycin, and cefotaxime. All Staphylococcus spp. displayed multidrug resistance (MDR) phenotype. Staphylococci isolates were highly resistant to fusidic acid, β-lactams, and tetracyclines. Amoxicillin-clavulanic acid, fosfomycin, and cephalosporines were ineffective against Enterobacteriaceae isolates. Ticarcillin, clavulanic acid, and colistin were ineffective against P. aeruginosa and Acinetobacter baumannii. Pan-drug-resistant (PDR) P. aeruginosa isolate was detected in the infected wound. Two lytic bacteriophages (vB_Pae_LP125 and vB_Pae_LS225) from the Podoviridea and Siphoviridea families were isolated from the horse manure. Both phages were stable across various temperatures and pH levels. In vitro tests showed significant lytic activity against a wide range of bacterial strains. The DNA genomes of all phages displayed distinctive restriction fragment length polymorphism. A bacteriophage cocktail (vB_Pae_LP125 and vB_Pae_LS225), when combined with gentamicin, improved wound healing in infected horses. There were significant differences (p < 0.05) in the wound closure % among the gentamicin group and phage cocktaoil+gentamicin groups on days 3, 5, 7, 10, and 14. This study highlights the widespread antibiotic resistance in bacteria infecting Arabian horses and posing significant challenges to equine infection management. Bacteriophage therapy shows promise as a potential treatment for wound infections.

抗菌素耐药性（Antimicrobial resistance）是一项重大的全球公共卫生问题，需要协调一致的应对措施。本研究首次针对引起阿拉伯马感染性疾病的细菌的流行率、抗菌素耐药表型，以及噬菌体疗法用于创伤治疗的潜力展开了调查。本研究共采集100份来自感染阿拉伯马的临床样本，这些马匹表现出呼吸系统疾病、腹泻、流产、创伤及眼部感染症状，采用直接样本多重聚合酶链式反应（multiplex PCR）与表型方法进行检测。对分离得到的菌株采用37种抗生素组合及肉汤微量稀释法进行药敏试验。从马粪便中分离得到噬菌体，并配制噬菌体鸡尾酒用于治疗阿拉伯马的感染性创伤。从呼吸道感染样本中分离得到的优势病原菌为马链球菌（Streptococcus equi）（17/29，58.6%），其次为肺炎克雷伯菌（Klebsiella pneumoniae）与铜绿假单胞菌（Pseudomonas aeruginosa）（各9/29，31.03%），以及大肠埃希菌（Escherichia coli）（7/29，24.13%）。金黄色葡萄球菌（Staphylococcus aureus）与羊棒杆菌马生物变种（Corynebacterium ovis biovar equi）是化脓性感染中最常分离到的细菌。所有分离得到的细菌均表现出多重抗生素耐药性。链球菌属（Streptococcus spp.）菌株呈现广泛耐药（XDR，extensive drug resistance）表型，对阿莫西林-克拉维酸（amoxicillin-clavulanic acid）、阿米卡星（amikacin）、卡那霉素（kanamycin）、链霉素（streptomycin）及头孢噻肟（cefotaxime）完全耐药。所有葡萄球菌属（Staphylococcus spp.）菌株均表现出多重耐药（MDR，multidrug resistance）表型，葡萄球菌分离株对夫西地酸（fusidic acid）、β-内酰胺类（β-lactams）及四环素类（tetracyclines）具有高度耐药性。阿莫西林-克拉维酸、磷霉素（fosfomycin）及头孢菌素类（cephalosporines）对肠杆菌科（Enterobacteriaceae）分离株无效。替卡西林（ticarcillin）、克拉维酸及黏菌素（colistin）对铜绿假单胞菌与鲍曼不动杆菌（Acinetobacter baumannii）无效。在感染创伤样本中检测到1株泛耐药（PDR，pan-drug-resistant）铜绿假单胞菌。从马粪便中分离得到2株属于短尾噬菌体科（Podoviridea）与长尾噬菌体科（Siphoviridea）的裂解性噬菌体vB_Pae_LP125和vB_Pae_LS225。两种噬菌体在多种温度与pH条件下均保持稳定。体外试验显示其对多种细菌菌株具有显著的裂解活性。所有噬菌体的DNA基因组均表现出独特的限制性片段长度多态性。将噬菌体鸡尾酒（vB_Pae_LP125与vB_Pae_LS225）与庆大霉素（gentamicin）联合使用，可改善感染马匹的创伤愈合情况。在第3、5、7、10及14天，庆大霉素组与噬菌体鸡尾酒+庆大霉素组的创伤闭合率存在显著差异（p < 0.05）。本研究凸显了感染阿拉伯马的细菌普遍存在抗菌素耐药性，这给马属动物感染的临床管理带来了重大挑战。噬菌体疗法有望成为感染性创伤的潜在治疗手段。