重金属协同选择环境细菌抗生素抗性及其机制研究进展

抗生素的长期滥用,引起环境细菌耐药性不断增强,加速了抗生素抗性基因(Antibiotic resistance genes,ARGs)在环境中的传播扩散。在重金属污染的环境中,细菌不仅具备重金属抗性,并且具备多种抗生素抗性,抗生素抗性基因的污染水平也随之升高。在介绍重金属与抗生素抗性最新研究进展的基础上,阐述了环境细菌的抗生素抗性、重金属抗性及其相关抗性机制,并着重论述重金属和抗生素协同选择环境细菌耐药性及其机制。     

Effects of environment on compensatory mutations to ameliorate costs of antibiotic resistance

Most types of antibiotic resistance impose a biological cost on bacterial fitness. These costs can be compensated, usually without loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a laboratory medium. Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a laboratory medium. This difference in mutation spectra was caused by either a growth condition-specific formation or selection of the compensated mutants. These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.     

中国和欧美国家养殖业抗生素使用负面问题及管理政策比较

畜禽养殖中抗生素的使用无疑推动了该产业的快速发展,但也引起了其耐药性威胁公共健康和养殖业可持续发展的担忧。本文系统梳理了兽用抗生素的使用,抗生素负面问题的显现、认知和管理政策的变化,分析了中国、美国、欧盟管理政策的差异及其背后的原因。本文认为,我国在推进农业现代化进程中：应长期关注养殖业中抗生素使用可能带来的负面问题,加快生产方式的绿色转型;应提升公众对抗生素耐药性的认识,引导公众参与遏制耐药性发展的行动;应考虑我国的发展目标,吸取美国因利益集团阻挠而反应缓慢的教训,综合考虑成本和收益,发挥我国的体制优势,找到政策上的最优解;支持、推动全球抗生素管理的国际协同行动。     

Multiple Antibiotic Resistance and Heavy Metal Resistance Profile of Bacteria Isolated from Giant Freshwater Prawn （Macrobrachium rosenbergii） Hatchery

In this article, antibiogram and heavy metal resistance profile of bacteria isolated from giant freshwater prawn （Macrobrachium rosenbergii） hatchery in Malaysia are described. Although giant freshwater prawn was introduced into Malaysia since the 1980s, there was no database information on antibiogram and heavy metal resistance profile of bacteria from giant freshwater prawn （34. rosenbergii） hatchery in Malaysia. Therefore, this study was carried out to determine the effectiveness of antibiotic and heavy metal resistance profile to control bacterial diseases in M. rosenbergii hatchery. The results can provide valuable information for local M. rosenbergii post-larval producer. Antibiotic sensitivity test was carried out by disk-diffusion method against 15 types of antibiotics as follows： oxolinic acid （2 μg）, ampicillin （10μg）, erythromycin （15μg）, furazolidone （15 μg）, lincomycin （15μg）, amoxicillin （25 μg）, colistin sulphate （25 μg）, doxycycline （30μg）, florfenicol （30 μg）, flumequine （30 lag）, nalidixic acid （30μg）, tetracycline （30μg）, oleandomycin （15μg）, fosfomycin （50μg）, and spiramycin （100 μg）, whereas heavy metal resistance profile of the present bacterial isolates was determined by 2-fold agar dilution technique. In this study, 5 types of bacteria were successfully isolated; they were Aeromonas spp. （n = 77）, Escherichia coli （n = 73）, Edwardsiella spp. （n = 62）, Salmonella spp. （n = 75）, and Vibrio spp. （n = 43）. The result showed that furazolidone was the most effective antibiotic to control the bacteria isolated in this study, approximately 89.7% of the bacterial isolates were sensitive to this antibiotic. Multiple antibiotic resistance （MAR） index indicated that the hatchery water source and M. rosenbergii post-larval and sediment tanks were at high-risk exposure to the tested antibiotic. Furthermore, all the tested heavy metals （Cd2＋, Cr6＋, Hg2＋, and Cu2＋） failed to inhibit the growth of the bacterial isolates. Therefore, it indicated that the water source of the hatchery is contaminated with both antibiotic residues and heavy metal.     

湛江东海岛对虾育苗场水体细菌的抗生素耐药性

利用K-B纸片扩散法研究分离自湛江东海岛6个对虾育苗场(分别编号为YF,JD,DC,HM,SD,XR)水体中的217株细菌菌株对12种抗生素的耐药性。研究结果表明,参试的12种抗生素之间的细菌耐药性差异显著(ANOVA,P0.05),6个育苗场内细菌对多粘菌素B、左氟沙星和环丙沙星高度敏感,对氨苄西林、甲氧苄胺嘧啶、复方新诺明和四环素则高度耐药;多重耐药菌株在每个育苗场中均有发现,52.07%的菌株呈现多重耐药性,未发现对参试的12种抗生素100%耐药的菌株。统计分析结果显示,尽管HM和XR育苗场的细菌耐药性比其他育苗场高,但6个对虾育苗场之间水体细菌的抗生素耐药性无显著差异(ANOVA,P0.05)。     

Darwinian evolution can follow only very few mutational paths to fitter proteins

Five point mutations in a particular beta-lactamase allele jointly increase bacterial resistance to a clinically important antibiotic by a factor of approximately 100,000. In principle, evolution to this high-resistance beta-lactamase might follow any of the 120 mutational trajectories linking these alleles. However, we demonstrate that 102 trajectories are inaccessible to Darwinian selection and that many of the remaining trajectories have negligible probabilities of realization, because four of these five mutations fail to increase drug resistance in some combinations. Pervasive biophysical pleiotropy within the beta-lactamase seems to be responsible, and because such pleiotropy appears to be a general property of missense mutations, we conclude that much protein evolution will be similarly constrained. This implies that the protein tape of life may be largely reproducible and even predictable.     

水产养殖环境中抗生素抗性基因污染及其研究进展

抗生素不仅能防治水产生物的细菌性疾病,还能促进养殖生物的生长,因此在水产养殖业中的使用极为广泛。中国作为世界水产养殖大国,近年来养殖生态环境所遭受的抗生素污染日益严重,并由此诱导产生了一种新型的环境污染物--抗生素抗性基因。此类污染物可通过基因水平的转移进入人体,最终危害人类的健康,正引起国内外的广泛关注。在了解水产养殖环境中抗生素使用情况的基础上,阐述了抗生素抗性基因的来源、作用机制、传播扩散机制及污染危害影响,并对未来抗生素抗性基因的研究方向进行了展望,以期引起相关研究人员的重视,为保护水产养殖环境和水产品质量安全提供依据。     

食品中阪崎克罗诺杆菌的药物敏感性及分子分型研究

克罗诺杆菌是一种革兰氏阴性菌,是配方粉中威胁婴幼儿健康的主要致病菌。近年来,乳制品及食品中常有克罗诺杆菌耐药株的报道,耐药株的出现给临床治疗带来巨大挑战。为探究PFGE型别与耐药表型之间的关联性,采用BD PhoenixTM-100全自动细菌鉴定及药敏系统对食品分离的68株阪崎克罗诺杆菌进行药敏检测,共选择19种抗生素,并采用PFGE对其进行分子分型。结果表明：4株阪崎克罗诺杆菌具有耐药性,耐药率为5.88%。其中,3株对头孢唑啉耐药,1株对四环素耐药。18株菌表现为中介,其中16株对头孢唑啉中介,2株对氯霉素中介。所有菌株对其余16种抗生素均敏感。68株阪崎克罗诺杆菌可分成58个PFGE带型,其中PT037、PT050、PT054、PT007、PT043及PT046带型分别含有4、3、3、2、2、2株菌,其余52个带型各含1株菌,带型较分散。3株头孢唑啉耐药菌株包含3个PFGE带型,18株中介菌株包含16个PFGE带型。检测的阪崎克罗诺杆菌食品分离株耐药率较低,未发现多重耐药菌株,且呈现出高度基因多态性,菌株PFGE带型与耐药性之间无明显相关性。     

High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection

The lungs of cystic fibrosis (CF) patients are chronically infected for years by one or a few lineages of Pseudomonas aeruginosa. These bacterial populations adapt to the highly compartmentalized and anatomically deteriorating lung environment of CF patients, as well as to the challenges of the immune defenses and antibiotic therapy. These selective conditions are precisely those that recent theoretical studies predict for the evolution of mechanisms that augment the rate of variation. Determination of spontaneous mutation rates in 128 P. aeruginosa isolates from 30 CF patients revealed that 36% of the patients were colonized by a hypermutable (mutator) strain that persisted for years in most patients. Mutator strains were not found in 75 non-CF patients acutely infected with P. aeruginosa. This investigation also reveals a link between high mutation rates in vivo and the evolution of antibiotic resistance.     

肉牛关节大肠杆菌的分离鉴定及药物敏感性分析

为了研究引起肉牛关节炎病原菌的种类及对抗生素的敏感性,采用细菌分离鉴定法、纸片扩散法对菌株进行鉴定和药物敏感性试验。结果表明：从2例病牛关节液中分离鉴定出2株大肠杆菌;用18 种抗生素进行药物敏感性试验,分离菌株对环丙沙星、丁胺卡那霉素、氟哌酸、庆大霉素、先锋必素5种药物敏感,对复达欣、卡那霉素、头孢呋肟3种药物中介,对羧苄青霉素、先锋霉素V、强力霉素、美满霉素、复方新诺明、红霉素、氯霉素、四环素、先锋霉素IV、氧哌嗪青霉素10 种药物产生耐药性。     

抗生素敏感性和消毒剂耐受性的潜在联系

食源性微生物的抗生素敏感性和消毒剂耐受性对食品安全和人类健康造成了巨大威胁,它直接或间接地导致了抗生素和消毒剂在治疗或消灭细菌的失败.该文综述了抗生素的多重耐药、消毒剂抗性机制及检测方法以及抗生素和消毒剂交叉耐药机制.未来的研究方向应该着重于对抗生素和消毒剂的使用提供专业指导以及研发出一套综合全面的分子生物学检测方法.     

噬菌体及其内溶素的应用研究进展

抗生素的滥用使越来越多的病原菌产生抗药性,因此寻求新的抗菌素已经变得极为迫切。噬菌体以及噬菌体编码的内溶素的发现和应用,为耐药性细菌的治疗和预防开辟了新的途径。文章综述了近几年噬菌体和内溶素的研究进展及其应用。     

一株分离自鸡肉样品的多重耐药大肠埃希菌的全基因组测序及耐药性研究

以分离自宁波市市售鸡肉中的一株大肠埃希菌ECCNB12-2为研究对象,使用微量肉汤稀释法进行最小抑菌浓度(MIC)测定,结果显示,该菌株对氨苄西林、庆大霉素、大观霉素、四环素、氟苯尼考、磺胺异恶唑、复方新诺明、头孢噻夫、恩诺沙星、氧氟沙星等10种抗生素耐药。采用第三代高通量测序技术对该菌株进行全基因组测序,随后对基因组完成图进行获得性耐药基因、毒力因子、质粒水平转移元件预测。菌株ECCNB12-2染色体基因组大小为5 539 489 bp,GC含量为50.37%,同时携带有4个质粒,大小分别为147 451 bp(pTB-nb1)、139 752 bp(pTB-nb2)、82 252 bp(pTB-nb3)、253 793 bp(pTB-nb4)。获得性耐药基因预测结果显示,染色体基因组、质粒pTB-nb1及质粒pTB-nb4上共携带有40个获得性耐药基因,同时菌株基因组检测出12个毒力因子。质粒水平转移元件预测结果显示,pTB-nb4质粒包含完整的水平转移系统,理论上具有高度的自主接合转移潜力。以上研究表明,分离自市售鸡肉样品的ECCNB12-2菌株是一株高风险的多重耐药菌株,反映了市售鸡肉中细菌耐药状况的严重性,相关研究结果为食源性细菌耐药安全风险评估提供了参考。     

TetR/AcrR转录因子Ms3469对耻垢分枝杆菌异烟肼抗药性的调控

以耻垢分枝杆菌(Mycobacterium smegmatis)为研究对象,通过在药物平板上筛选转录因子超表达文库,发现MSMEG_4369(Ms3469)影响耻垢分枝杆菌(M.smegmatis)对抗结核一线药物异烟肼(Isoniazid,INH)的敏感性。进一步研究发现超表达Ms3469能增强耻垢分枝杆菌的INH抗性;基因保守结构域分析表明Ms3469编码一个TetR/AcrR家族的转录调节蛋白;凝胶迁移率阻滞实验和细菌单杂交实验证明Ms3469能够与自身启动子直接特异结合。     

Antibiogram and heavy metal resistance of pathogenic bacteria isolated from moribund cage cultured silver catfish (<Emphasis Type="Italic">Pangasius sutchi</Emphasis>) and red hybrid tilapia (<Emphasis Type="Italic">Tilapia</Emphasis> sp.)

Antibiogram and heavy metal resistance patterns of pathogenic bacteria isolated from moribund cage cultured silver catfish (Pangasius sutchi) and red hybrid tilapia (Tilapia sp.) from Sungai Manir, Terengganu, Malaysia were studied and characterized. Sungai Manir is one of the famous rivers in Terengganu for its wide variety of cage cultured freshwater fish. However, to date, the baseline information of antibiogram and heavy metal resistance patterns of the pathogenic bacteria attacking the freshwater fish cultured in Sungai Manir is still lacking. Therefore, this study was carried out, which may be useful for fish farmers as a guideline for fish prophylactic and treatment purposes. Furthermore, present studies also provide information on the safety level of consuming freshwater fish produced from Sungai Manir. In the present study, bacteria were isolated from 100 fish of each moribund silver catfish and red hybrid tilapia using seven media including tryptic soy agar (TSA), Mac Conkey, thiosulphate citrate bile salt (TCBS), eosin methylene blue (EMB), glutamate starch pseudomonas (GSP), xylose lysine deoxycholate (XLD) and Baird Parker media. Identification of bacteria was carried out using conventional biochemical tests and confirmed by commercial bacterial identification kit. Antibiogram of the bacterial isolates against 18 antibiotics; oxolinic acid (2 μg), ampicillin (10 μg), erythromycin (15 μg), furazolidone (15 μg), lincomycin (15 μg), oleandomycin (15 μg), amoxicillin (25 μg), colistin sulphate (25 μg), sulphamethoxazole (25 μg), chloramphenicol (30 μg), doxycycline (30 μg), florfenicol (30 μg), flumequine (30 μg), kanamycin (30 μg), nalidixic acid (30 μg), tetracycline (30 μg), nitrofurantoin (50 μg) and spiramycin (100 μg) was carried out using disk diffusion method, whereas heavy metal resistance patterns (Hg²⁺, Cd²⁺, Cr^{6 +} and Cu²⁺) of the bacterial isolates was determined through twofold agar dilution method. The results showed that the percentage of sensitivity case of the 120 bacterial isolates to the tested antibiotics was 62.7%. This was followed by resistance (26.9%) and intermediary sensitive (10.4%) cases. In terms of the heavy metal resistance patterns, all bacterial isolates were resistant to Hg²⁺ and Cr^{6 +}. However, only 27.8% and 16.7% of the bacterial isolates were sensitive to Cu²⁺ and Cd²⁺, respectively. The multiple antibiotic resistance (MAR) indices indicated that the cage cultured silver catfish and red hybrid tilapia were under high exposure to the tested antibiotic. Overall, the results of the present studies showed that Sungai Manir may be polluted with heavy metal and antibiotic residues.     

TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification

Host protection from infection relies on the recognition of pathogens by innate pattern-recognition receptors such as Toll-like receptors (TLRs). Here, we show that the orphan receptor TLR13 in mice recognizes a conserved 23S ribosomal RNA (rRNA) sequence that is the binding site of macrolide, lincosamide, and streptogramin group (MLS) antibiotics (including erythromycin) in bacteria. Notably, 23S rRNA from clinical isolates of erythromycin-resistant Staphylococcus aureus and synthetic oligoribonucleotides carrying methylated adenosine or a guanosine mimicking a MLS resistance-causing modification failed to stimulate TLR13. Thus, our results reveal both a natural TLR13 ligand and specific mechanisms of antibiotic resistance as potent bacterial immune evasion strategy, avoiding recognition via TLR13.     

The shared antibiotic resistome of soil bacteria and human pathogens

Soil microbiota represent one of the ancient evolutionary origins of antibiotic resistance and have been proposed as a reservoir of resistance genes available for exchange with clinical pathogens. Using a high-throughput functional metagenomic approach in conjunction with a pipeline for the de novo assembly of short-read sequence data from functional selections (termed PARFuMS), we provide evidence for recent exchange of antibiotic resistance genes between environmental bacteria and clinical pathogens. We describe multidrug-resistant soil bacteria containing resistance cassettes against five classes of antibiotics (β-lactams, aminoglycosides, amphenicols, sulfonamides, and tetracyclines) that have perfect nucleotide identity to genes from diverse human pathogens. This identity encompasses noncoding regions as well as multiple mobilization sequences, offering not only evidence of lateral exchange but also a mechanism by which antibiotic resistance disseminates.     

圈养高山兀鹫肠道微生物分离、鉴定与抗生素耐药性分析

高山兀鹫(Gyps himalayensis)是国家Ⅱ级保护鸟类,被世界自然保护联盟(IUCN)列入濒危物种红色名录。本试验对圈养高山兀鹫粪便细菌进行分离与培养、16S rRNA基因扩增与测序、细菌耐药性测试。经鉴定,分离出的菌株隶属于2门、8属、9种,分别是弗氏柠檬酸杆菌(Citrobacter freundii)、太平洋芽孢杆菌(Bacillus pacificus)、粪肠球菌(Enterococcus faecalis)、蜂房哈夫尼亚菌(Hafnia alvei)、豪氏变形杆菌(Proteus hauseri)、普通变形杆菌(Proteus vulgaris)、腐败希瓦氏菌(Shewanella putrefaciens)、运动肉杆菌(Carnobacterium mobile)、福氏志贺氏菌(Shigella flexneri)。这9种菌株对3到11种不等的抗生素表现出耐药性。其中,运动肉杆菌、粪肠球菌和普通变形杆菌可耐受大多数的抗生素。福氏志贺氏菌可耐受的抗生素种类较少。本研究为圈养高山兀鹫的肠道微生物及其抗生素耐药性提供了相关数据,进一步为高山兀鹫的保护性人工养殖提供参考。     

The ins and outs of DNA transfer in bacteria

Transformation and conjugation permit the passage of DNA through the bacterial membranes and represent dominant modes for the transfer of genetic information between bacterial cells or between bacterial and eukaryotic cells. As such, they are responsible for the spread of fitness-enhancing traits, including antibiotic resistance. Both processes usually involve the recognition of double-stranded DNA, followed by the transfer of single strands. Elaborate molecular machines are responsible for negotiating the passage of macromolecular DNA through the layers of the cell surface. All or nearly all the machine components involved in transformation and conjugation have been identified, and here we present models for their roles in DNA transport.