养殖场空气中细菌分布及耐药性研究
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摘要
空气是人和动物赖以生存的重要环境因素,一些病原菌以空气为媒介(微生物气溶胶)进行扩散传播,不仅污染了养殖环境,甚至还可引起某些传染病的暴发流行,给养殖业造成巨大的经济损失。空气微生物的种类和含量已成为畜禽舍环境质量的重要指标,直接关系到饲养人员、养殖动物的健康以及生产性能的发挥。以往有关动物舍空气微生物的监测主要针对某种特定的致病菌或是某一类细菌,往往不能了解到空气中菌群分布的整体状况。此外,在对菌群的定性研究中,采用的方法大多依赖于传统的形态学、血清学、生化反应、分子生物学等方法,虽然这些技术在细菌鉴定方面各具特色,但均无法满足对空气微生物进行准确、快速、高通量的检测要求。
近年来随着抗生素的大量使用,细菌耐药性、药物残留等问题日趋严重,抗生素和耐药株已对养殖环境造成了不同程度的污染,但有关空气中细菌耐药情况的调查相对较少,目前我国尚缺少此方面的研究资料。针对以上问题,本课题选取了长春市五家规模化养猪场,开展了如下研究,旨在对预防养殖场畜禽疾病、合理使用抗生素、改善饲养环境等方面提供理论依据。
1.基于MALDI-TOF质谱技术的细菌检测方法的建立。利用基质辅助激光解析电离飞行时间质谱仪(Matrix-Assistant Laser Desorption /Ionization Time of Flight Mass Spectrometry, MALDI-TOF MS)建立了一套基于该技术的细菌检测方法(包括样品前处理方法、检测范围、激光能量等),并研究了培养基、培养时间、样品保存时间等实验条件的改变对细菌质谱图的影响。同时以16SrDNA测序法作为参照方法,利用MALDI-TOF质谱技术检测了50株分离株,评估其检测能力。结果显示,质谱检测方法重复性好,不受培养基、培养时间、不同型号仪器等实验条件的影响,所获图谱质量较好、特征峰数目多、信号强度高、离子峰的分布模式稳定。与16SrDNA测序法相比较,结果显示二者具有极高的一致性。以上结果表明,MALDI-TOF质谱技术具有可靠、快速、高通量检测细菌的能力。
2.养殖场空气中细菌种类及耐药性整体分布情况。本研究采集了长春地区五家养猪场的空气样本,培养基法对样本进行分离培养,并利用上述建立的质谱技术检测法对空气分离株进行鉴定。结果显示,养殖场空气中细菌种类多为环境正常菌群及条件性病原菌。其中革兰氏阳性菌明显居多,占细菌总数的78.1%,以凝固酶阴性葡萄球菌为优势菌群,占据62.7%,其次为芽孢杆菌、气球菌属和罗氏菌属。革兰氏阴性菌中以肠杆菌属数量最多,不动杆菌属、假单胞菌属分别位居二、三位。
利用Vitek32全自动微生物分析仪、药敏纸片等表型法对养殖场空气分离株进行了药物敏感性检测。结果显示,耐药株检出率较高,主要集中在对红霉素、克林霉素、四环素这些常用于饲料中促生长剂的药物,部分菌株表现出对青霉素类、喹诺酮类等临床上人兽共用药物的耐受性。研究表明,作为优势菌群的凝固酶阴性葡萄球菌(CNS)耐药情况非常严重,尤其是耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)检出率高达65.9%,并伴随着多重耐药现象出现。此外,发现一株鼠鼻罗氏菌(Rothia nasimurium)对除万古霉素外的所有抗生素均表现出高水平耐药,这在以往有关罗氏菌的文献中未见报道。
3.凝固酶阴性葡萄球菌耐药基因检测。为了深入了解当前长春地区养殖环境中耐药性的流行特点,本研究以CNS为研究对象,针对β内酰胺类、大环内酯类和四环素这三类耐药率较高的抗生素,采用PCR法对其相关耐药基因进行检测。结果显示mecA检出率为58.0%,部分菌株表现为苯唑西林耐药,mecA阴性结果;ermC(69.6%)是阳性率最高的大环内酯类耐药基因,其次是msrA(15.7%)和ermB(14.8%),未检测到ermA基因;四环素类耐药基因tetK(62.7%)检出率明显高于tetM(25.4%)。
4.高水平多重耐药株鼠鼻罗氏菌的比较蛋白质组学研究。为了解鼠鼻罗氏菌产生耐药性的机制,本研究利用双向电泳和质谱技术比较了鼠鼻罗氏菌耐药株和敏感株全蛋白的变化差异,共筛选并鉴定出18个蛋白,并通过半定量RT-PCR的方法在mRNA水平上对18个差异蛋白进行了验证,结果有4种蛋白的基因在转录水平上表现出与双向电泳结果的一致性,经数据库查询及文献分析这4种蛋白很可能与鼠鼻罗氏菌的耐药性密切相关。
通过以上研究结果,得出以下结论:
1.本研究建立的基于质谱技术的检测方法能够快速、准确和高通量的检测鉴定细菌。
2.明确了调查区域养殖场空气中以凝固酶阴性葡萄球菌为优势菌群的细菌分布特点,了解到被调查养殖场空气分离株对β内酰胺类、大环内酯类、四环素类等饲料和临床中常用抗生素具有不同程度的耐药性。
3.明确了调查区域养殖场空气中优势菌群(CNS)耐药基因的分布特点:mecA,ermC,tetK分别与环境中CNS对β内酰胺类、大环内酯类及四环素耐药性的产生密切相关。
4.利用蛋白质组学及半定量RT-PCR筛选并鉴定到的4种差异蛋白,很可能与鼠鼻罗氏菌的多重耐药性密切相关,这将有助于发现和探究这些蛋白的新功能,并为明确该菌的耐药机制奠定理论基础。
Air is one of the most important environmental factors for human and animals. The airborne microorganisms can cause serious air pollution and induce prevalence of some infective diseases, as well as enormous economic lost. Recently, due to the large antibiotics usage, it has become more serious in resistant bacteria and antibiotics residues. Although, the antibiotics and resistance in bacteria have caused pollution of farm environments, there are limited investigations about airborne bacterial resistance and it has not been much concerned in our country. In the past, the detection about airborne microorganisms was often based on some special pathogens or species, but it couldn’t understand the whole distribution of airborne bacteria. Besides that, the methods for bacterial identification were mainly dependent on morphology, biochemical characterization, molecular biology and so on. But they couldn’t satisfy the demands of speed and high-throughput screening. Based on above issues, we have carried out following study in order to provide guidance on the prevention of farm animal diseases, improvement of breeding environments, maintenance of animal health and increasing of production performance.
1. The establishment of bacterial detection based on MALDI-TOF MS:
A method for bacterial identification was established by using MALDI-TOF MS, which included sample preparation, detection extent, and laser power. This project researched the influences of the mass spectrum when detecting bacteria by MALDI-TOF MS in several experimental aspects such as cultivation time, cultivation media and conservation time. To evaluate the capability of MALDI-TOF MS, 50 clinical bacterial strains were analyzed. As a reference method for species designation, 16S rRNA gene sequencing was applied. The results showed good reproducibility for mass spectrum without affected by cultivation media, cultivation time or different instruments. The obtained mass spectrum had good quality because of many characteristic peaks, high intensive signals and stable distribution pattern of ion peaks. There was a high accordance with 16S rRNA gene sequencing and MS. It indicated that MALDI-TOF MS-based species identification of bacteria could provide reliable, rapid and high-throughput results.
2. The global distribution of species and resistance of airborne bacteria in farms:
In this study, we isolated bacteria from air samples of five swine farms in Changchun area and detected them by MALDI-TOF MS. It displayed that there were almost normal environmental bacterial population and opportunity pathogens in the air. Among these, 78.1% were gram-positive bacteria and coagulase negative Staphylococci were predominant, as followed Bacillus, Aerococcus and Rothia. The Enterobacteriaceae were the highest bacteria population in gram-negative bacteria, and the Acinetobacter, Pseudomonas ranked the second and third, respectively.
The results of Vitek32 Automated Microbiology System and Kirby-Bauer(K-B) showed that there was a high incidence of resistant bacteria, which mainly focused on erythromycin, clindamycin and tetracycline. Parts of bacteria presented resistance on penicillins and quinolones which used in human and animal clinical treatment. It demonstrated that as the dominant flora of coagulase-negative Staphylococci (CNS) drug resistance was very serious, especially methicillin-resistant coagulase-negative Staphylococci (MRCNS), the detective rate was 65.9% and most of them were multi-drug resistant. This study also found a strain of Rothia nasimurium (No.91) which represented high-level resistance to all tested antibiotics except to vancomycin. It has never been seen in all airborne bacteria and never reported in Rothia previously.
3. Detection of resistance genes in coagulase-negative Staphylococci:
To understand the distribution and molecular mechanisms of CNS resistant in the current farming environment in Jilin area, the PCR method was used to detect resistance genes related withβ-lactams, macrolides and tetracycline which had high resistant rates. It showed that 58.0% of mecA gene was detected and some strains showed oxacillin resistant, but were negative results of mecA. ermC (69.6%) was the highest positive rate of macrolide resistance gene, followed by msrA (15.7%), ermB (14.8%). The ermA gene was not detected. The rate of tetracycline resistant gene tetK (62.7%) was higher than tetM (25.4%).
4. Comparative proteomics of high-level multi-drug resistant Rothia nasimurium:
To understand resistant mechanism of Rothia nasimurium, this project utilized two-dimensional electrophoresis and mass spectrometry to compare the differences of proteins between drug-resistant strain (No.91) and sensitive strain. There were a total of 18 different proteins to be screen and identify. The proteins had been verified in mRNA levels by semi-quantitative RT-PCR method. Results demonstrated that there were four protein genes at the transcription level in accordance with the two-dimensional electrophoresis. It also implicated that there would be a close relationship between the four proteins and drug resistance.
As per above results, our conclusions are as fllows:
1. The established method for bacterial detection based on MALDI-TOF MS possesed more advantages that could rapid, accurate and high-throughput to detect and identify bacteria. It could also be used in airborne microorganism’s research.
2. It was clear that the distribution of airborne bacteria was the CNS as the dominant flora in the investigated farms. It was understood that the airborne bacteria displayed different degree drug resistance toβ-lactams, macrolides, tetracyclines and other antibiotics which used in feed and clinical treatment.
3. It was clear that the prevalence of air-borne CNS resistance genes in investigated farms: mecA, ermC, tetK were closely related to resistance ofβ-lactam, macrolide and tetracycline, respectively.
4. The four different proteins screened and identified by 2D were probably associated with antibiotic resistance of Rothia nasimurium. It would help to discover and explore the new functions of these proteins and provide theoretical basis for specifying bacteria resistant mechanism.
近年来随着抗生素的大量使用,细菌耐药性、药物残留等问题日趋严重,抗生素和耐药株已对养殖环境造成了不同程度的污染,但有关空气中细菌耐药情况的调查相对较少,目前我国尚缺少此方面的研究资料。针对以上问题,本课题选取了长春市五家规模化养猪场,开展了如下研究,旨在对预防养殖场畜禽疾病、合理使用抗生素、改善饲养环境等方面提供理论依据。
1.基于MALDI-TOF质谱技术的细菌检测方法的建立。利用基质辅助激光解析电离飞行时间质谱仪(Matrix-Assistant Laser Desorption /Ionization Time of Flight Mass Spectrometry, MALDI-TOF MS)建立了一套基于该技术的细菌检测方法(包括样品前处理方法、检测范围、激光能量等),并研究了培养基、培养时间、样品保存时间等实验条件的改变对细菌质谱图的影响。同时以16SrDNA测序法作为参照方法,利用MALDI-TOF质谱技术检测了50株分离株,评估其检测能力。结果显示,质谱检测方法重复性好,不受培养基、培养时间、不同型号仪器等实验条件的影响,所获图谱质量较好、特征峰数目多、信号强度高、离子峰的分布模式稳定。与16SrDNA测序法相比较,结果显示二者具有极高的一致性。以上结果表明,MALDI-TOF质谱技术具有可靠、快速、高通量检测细菌的能力。
2.养殖场空气中细菌种类及耐药性整体分布情况。本研究采集了长春地区五家养猪场的空气样本,培养基法对样本进行分离培养,并利用上述建立的质谱技术检测法对空气分离株进行鉴定。结果显示,养殖场空气中细菌种类多为环境正常菌群及条件性病原菌。其中革兰氏阳性菌明显居多,占细菌总数的78.1%,以凝固酶阴性葡萄球菌为优势菌群,占据62.7%,其次为芽孢杆菌、气球菌属和罗氏菌属。革兰氏阴性菌中以肠杆菌属数量最多,不动杆菌属、假单胞菌属分别位居二、三位。
利用Vitek32全自动微生物分析仪、药敏纸片等表型法对养殖场空气分离株进行了药物敏感性检测。结果显示,耐药株检出率较高,主要集中在对红霉素、克林霉素、四环素这些常用于饲料中促生长剂的药物,部分菌株表现出对青霉素类、喹诺酮类等临床上人兽共用药物的耐受性。研究表明,作为优势菌群的凝固酶阴性葡萄球菌(CNS)耐药情况非常严重,尤其是耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)检出率高达65.9%,并伴随着多重耐药现象出现。此外,发现一株鼠鼻罗氏菌(Rothia nasimurium)对除万古霉素外的所有抗生素均表现出高水平耐药,这在以往有关罗氏菌的文献中未见报道。
3.凝固酶阴性葡萄球菌耐药基因检测。为了深入了解当前长春地区养殖环境中耐药性的流行特点,本研究以CNS为研究对象,针对β内酰胺类、大环内酯类和四环素这三类耐药率较高的抗生素,采用PCR法对其相关耐药基因进行检测。结果显示mecA检出率为58.0%,部分菌株表现为苯唑西林耐药,mecA阴性结果;ermC(69.6%)是阳性率最高的大环内酯类耐药基因,其次是msrA(15.7%)和ermB(14.8%),未检测到ermA基因;四环素类耐药基因tetK(62.7%)检出率明显高于tetM(25.4%)。
4.高水平多重耐药株鼠鼻罗氏菌的比较蛋白质组学研究。为了解鼠鼻罗氏菌产生耐药性的机制,本研究利用双向电泳和质谱技术比较了鼠鼻罗氏菌耐药株和敏感株全蛋白的变化差异,共筛选并鉴定出18个蛋白,并通过半定量RT-PCR的方法在mRNA水平上对18个差异蛋白进行了验证,结果有4种蛋白的基因在转录水平上表现出与双向电泳结果的一致性,经数据库查询及文献分析这4种蛋白很可能与鼠鼻罗氏菌的耐药性密切相关。
通过以上研究结果,得出以下结论:
1.本研究建立的基于质谱技术的检测方法能够快速、准确和高通量的检测鉴定细菌。
2.明确了调查区域养殖场空气中以凝固酶阴性葡萄球菌为优势菌群的细菌分布特点,了解到被调查养殖场空气分离株对β内酰胺类、大环内酯类、四环素类等饲料和临床中常用抗生素具有不同程度的耐药性。
3.明确了调查区域养殖场空气中优势菌群(CNS)耐药基因的分布特点:mecA,ermC,tetK分别与环境中CNS对β内酰胺类、大环内酯类及四环素耐药性的产生密切相关。
4.利用蛋白质组学及半定量RT-PCR筛选并鉴定到的4种差异蛋白,很可能与鼠鼻罗氏菌的多重耐药性密切相关,这将有助于发现和探究这些蛋白的新功能,并为明确该菌的耐药机制奠定理论基础。
Air is one of the most important environmental factors for human and animals. The airborne microorganisms can cause serious air pollution and induce prevalence of some infective diseases, as well as enormous economic lost. Recently, due to the large antibiotics usage, it has become more serious in resistant bacteria and antibiotics residues. Although, the antibiotics and resistance in bacteria have caused pollution of farm environments, there are limited investigations about airborne bacterial resistance and it has not been much concerned in our country. In the past, the detection about airborne microorganisms was often based on some special pathogens or species, but it couldn’t understand the whole distribution of airborne bacteria. Besides that, the methods for bacterial identification were mainly dependent on morphology, biochemical characterization, molecular biology and so on. But they couldn’t satisfy the demands of speed and high-throughput screening. Based on above issues, we have carried out following study in order to provide guidance on the prevention of farm animal diseases, improvement of breeding environments, maintenance of animal health and increasing of production performance.
1. The establishment of bacterial detection based on MALDI-TOF MS:
A method for bacterial identification was established by using MALDI-TOF MS, which included sample preparation, detection extent, and laser power. This project researched the influences of the mass spectrum when detecting bacteria by MALDI-TOF MS in several experimental aspects such as cultivation time, cultivation media and conservation time. To evaluate the capability of MALDI-TOF MS, 50 clinical bacterial strains were analyzed. As a reference method for species designation, 16S rRNA gene sequencing was applied. The results showed good reproducibility for mass spectrum without affected by cultivation media, cultivation time or different instruments. The obtained mass spectrum had good quality because of many characteristic peaks, high intensive signals and stable distribution pattern of ion peaks. There was a high accordance with 16S rRNA gene sequencing and MS. It indicated that MALDI-TOF MS-based species identification of bacteria could provide reliable, rapid and high-throughput results.
2. The global distribution of species and resistance of airborne bacteria in farms:
In this study, we isolated bacteria from air samples of five swine farms in Changchun area and detected them by MALDI-TOF MS. It displayed that there were almost normal environmental bacterial population and opportunity pathogens in the air. Among these, 78.1% were gram-positive bacteria and coagulase negative Staphylococci were predominant, as followed Bacillus, Aerococcus and Rothia. The Enterobacteriaceae were the highest bacteria population in gram-negative bacteria, and the Acinetobacter, Pseudomonas ranked the second and third, respectively.
The results of Vitek32 Automated Microbiology System and Kirby-Bauer(K-B) showed that there was a high incidence of resistant bacteria, which mainly focused on erythromycin, clindamycin and tetracycline. Parts of bacteria presented resistance on penicillins and quinolones which used in human and animal clinical treatment. It demonstrated that as the dominant flora of coagulase-negative Staphylococci (CNS) drug resistance was very serious, especially methicillin-resistant coagulase-negative Staphylococci (MRCNS), the detective rate was 65.9% and most of them were multi-drug resistant. This study also found a strain of Rothia nasimurium (No.91) which represented high-level resistance to all tested antibiotics except to vancomycin. It has never been seen in all airborne bacteria and never reported in Rothia previously.
3. Detection of resistance genes in coagulase-negative Staphylococci:
To understand the distribution and molecular mechanisms of CNS resistant in the current farming environment in Jilin area, the PCR method was used to detect resistance genes related withβ-lactams, macrolides and tetracycline which had high resistant rates. It showed that 58.0% of mecA gene was detected and some strains showed oxacillin resistant, but were negative results of mecA. ermC (69.6%) was the highest positive rate of macrolide resistance gene, followed by msrA (15.7%), ermB (14.8%). The ermA gene was not detected. The rate of tetracycline resistant gene tetK (62.7%) was higher than tetM (25.4%).
4. Comparative proteomics of high-level multi-drug resistant Rothia nasimurium:
To understand resistant mechanism of Rothia nasimurium, this project utilized two-dimensional electrophoresis and mass spectrometry to compare the differences of proteins between drug-resistant strain (No.91) and sensitive strain. There were a total of 18 different proteins to be screen and identify. The proteins had been verified in mRNA levels by semi-quantitative RT-PCR method. Results demonstrated that there were four protein genes at the transcription level in accordance with the two-dimensional electrophoresis. It also implicated that there would be a close relationship between the four proteins and drug resistance.
As per above results, our conclusions are as fllows:
1. The established method for bacterial detection based on MALDI-TOF MS possesed more advantages that could rapid, accurate and high-throughput to detect and identify bacteria. It could also be used in airborne microorganism’s research.
2. It was clear that the distribution of airborne bacteria was the CNS as the dominant flora in the investigated farms. It was understood that the airborne bacteria displayed different degree drug resistance toβ-lactams, macrolides, tetracyclines and other antibiotics which used in feed and clinical treatment.
3. It was clear that the prevalence of air-borne CNS resistance genes in investigated farms: mecA, ermC, tetK were closely related to resistance ofβ-lactam, macrolide and tetracycline, respectively.
4. The four different proteins screened and identified by 2D were probably associated with antibiotic resistance of Rothia nasimurium. It would help to discover and explore the new functions of these proteins and provide theoretical basis for specifying bacteria resistant mechanism.
引文
[1] Noah Fierer, Zongzhi Liu, Mari Rodriguez-Hernandez, et al. Short-Term Temporal Variability in airborne bacterial and fungal populations[J]. Applied and Environmental Microbiology, 2008, 74(1):200-207.
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