硫酸铜溶液诱导对耐甲氧西林金黄色葡萄球菌抗菌药物抗性效应的初步研究

doi:10.11843/j.issn.0366-6964.2020.04.021

摘要/Abstract

摘要： 旨在探究硫酸铜溶液连续诱导对耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）抗菌药物抗性及相关基因表达的影响。采用微量肉汤稀释法测定经硫酸铜诱导前后MRSA菌株（MR-YB1224、MR-YS3和MR-P318）对硫酸铜溶液及β-内酰胺类（氨苄西林、苯唑西林和头孢西丁）、氟喹诺酮类（氧氟沙星）、大环内酯类（罗红霉素）和氨基糖苷类（链霉素）等抗菌药物的最小抑菌浓度（minimum inhibitory concentration，MIC）；采用荧光定量PCR方法测定硫酸铜诱导对菌株抗重金属基因（copA和arsB）和耐药基因（mecA，norA，ermB和aac6'/aph2″）表达的影响。连续诱导7 d后，MRSA菌株对硫酸铜溶液的MIC值均增加；特别是对β-内酰胺类抗菌药物的MIC值增加显著（P<0.05）；MRSA菌株对6种抗菌药物MIC值变化大小依次为苯唑西林 > 氨苄西林 > 头孢西丁 > 氧氟沙星 > 链霉素 > 罗红霉素。另外，连续诱导后，MRSA菌株的抗重金属基因和相关耐药基因的表达均显著上调（P<0.05）。本试验结果表明，硫酸铜溶液诱导对MRSA菌株抗菌药物抗性效应具有较强的协同效应。

关键词: 金黄色葡萄球菌, 硫酸铜溶液, 诱导, 抗菌药物抗性, 抗重金属基因, 荧光定量PCR

Abstract: The study aimed to investigate the effects of copper sulfate solution continuous induction on the antibiotic resistance ability and the expression of related resistance genes of methicillin-resistant Staphylococcus aureus(MRSA) isolates. The minimum inhibitory concentrations(MICs) of MRSA strains (MR-YB1224, MR-YS3 and MR-P318) to copper sulfate, β-lactam(including ampicillin, oxacillin and cefoxitin), fluoroquinolones(including ofloxacin), macrolides(including roxithromycin) and aminoglycosides(including streptomycin) were determined using the standard broth microdilution method. A quantitative real time-PCR was applied to monitor relative expression levels of anti-heavy metal genes (copA, arsB) and antibiotic resistance genes (mecA, norA, ermB, aac6'/aph2″). After continuous induction by copper sulfate solution for 7 days, the MIC values to copper sulfate solution increased for three MRSA strains; especially the MIC values to β-lactam antibiotics significantly increased after induction (P<0.05). The change of MIC values to the different antibiotics were as follows:oxacillin > ampicillin > cefoxitin > ofloxacin > streptomycin > roxithromycin. In addition, the expression of the anti-heavy metal genes and related antibiotic resistance genes for the three MRSA strains were significantly up-regulated (P<0.05). The results show that copper sulfate solution has a strong synergistic effect on the antibiotic resistance of MRSA strains.

Key words: Staphylococcus aureus, copper sulfate solution, induction, antibiotic resistance, anti-heavy metal genes, RT-PCR

中图分类号:

S825.611

罗梦幽, 汤承, 赵燕英, 陈娟, 唐俊妮. 硫酸铜溶液诱导对耐甲氧西林金黄色葡萄球菌抗菌药物抗性效应的初步研究[J]. 畜牧兽医学报, 2020, 51(4): 841-850.

LUO Mengyou, TANG Cheng, ZHAO Yanying, CHEN Juan, TANG Junni. The Study of Copper Sulfate Solution Induction on the Resistance Effect of Methicillin-resistant Staphylococcus aureus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4): 841-850.

参考文献 36

[1]	WERTHEIM H F,MELLES D C,VOS M C,et al.The role of nasal carriage in Staphylococcus aureus infections[J].Lancet Infect Dis,2005,5(12):751-762.
[2]	SUAYA J A,MERA R M,CASSIDY A,et al.Incidence and cost of hospitalizations associated with Staphylococcus aureus skin and soft tissue infections in the United States from 2001 through 2009[J].BMC Infect Dis,2014,14:296.
[3]	CAHILL T J,BADDOUR L M,HABIB G,et al.Challenges in infective endocarditis[J].J Am Coll Cardiol,2017,69(3):325-344.
[4]	LAKHUNDI S,ZHANG K Y.Methicillin-resistant Staphylococcus aureus:molecular characterization,evolution,and epidemiology[J].Clin Microbiol Rev,2018,31(4):e00020-18.
[5]	SMITH T C.Livestock-associated Staphylococcus aureus:the united states experience[J].PLoS Pathog,2015,11(2):e1004564.
[6]	VOSS A,LOEFFEN F,BAKKER J,et al.Methicillin-resistant Staphylococcus aureus in pig farming[J].Emerg Infect Dis,2005,11(12):1965-1966.
[7]	ESPIGARES E,ROLDAN E M,ESPIGARES M,et al.Phenotypic resistance to disinfectants and antibiotics in methicillin-resistant Staphylococcus aureus strains isolated from pigs[J].Zoonoses Public Health,2017,64(4):272-280.
[8]	FISCHBACH M A,WALSH C T.Antibiotics for emerging pathogens[J].Science,2009,325(5944):1089-1093.
[9]	张行,杨峰,李新圃,等.牛源耐甲氧西林金黄色葡萄球菌耐药性与外排泵相关基因的检测及相关性研究[J].畜牧兽医学报,2019,50(11):2326-2332.ZHANG H,YANG F,LI X P,et al.Study on relationship between antimicrobial resistance and efflux pump related genes of methicillin-resistant Staphylococcus aureus in dairy cow[J].Acta Veterinaria et Zootechnica Sinica,2019,50(11):2326-2332.(in Chinese)
[10]	YU Z Y,GUNN L,WALL P,et al.Antimicrobial resistance and its association with tolerance to heavy metals in agriculture production[J].Food Microbiol,2017,64:23-32.
[11]	ARGUDÍN M A,HOEFER A,BUTAYE P.Heavy metal resistance in bacteria from animals[J].Res Vet Sci,2019,122:132-147.
[12]	MURPHY D,RICCI A,AUCE Z,et al.EMA and EFSA joint scientific opinion on measures to reduce the need to use antimicrobial agents in animal husbandry in the European Union,and the resulting impacts on food safety (RONAFA)[J].EFSA J,2017,15(1):4666.
[13]	王小垒,李凤姿,何家乐,等.环境微生物抗生素与重金属抗性研究进展[J].环境科技,2019,32(1):59-62.WANG X L,LI F Z,HE J L,et al.The research progress of environmental microbial antibiotic and heavy metal resistance[J].Environmental Science and Technology,2019,32(1):59-62.(in Chinese)
[14]	HOBMAN J L,CROSSMAN L C.Bacterial antimicrobial metal ion resistance[J].J Med Microbiol,2015,64(5):471-497.
[15]	UG A,CEYLAN Ö.Occurrence of resistance to antibiotics,metals,and plasmids in clinical strains of Staphylococcus spp[J].Arch Med Res,2003,34(2):130-136.
[16]	ZHAO Y,SU J Q,AN X L,et al.Feed additives shift gut microbiota and enrich antibiotic resistance in swine gut[J].Sci Total Environ,2018,621:1224-1232.
[17]	SLIFIERZ M J,FRIENDSHIP R,WEESE J S.Zinc oxide therapy increases prevalence and persistence of methicillin-resistant Staphylococcus aureus in pigs:a randomized controlled trial[J].Zoonoses Public Health,2015,62(4):301-308.
[18]	AMACHAWADI R G,SCOTT H M,NITIKANCHANA S,et al.Nasal carriage of mecA-positive methicillin-resistant Staphylococcus aureus in pigs exhibits dose-response to zinc supplementation[J].Foodborne Pathog Dis,2015,12(2):159-163.
[19]	BEDNORZ C,OELGESCHLÄGER K,KINNEMANN B,et al.The broader context of antibiotic resistance:zinc feed supplementation of piglets increases the proportion of multi-resistant Escherichia coli in vivo[J].Int J Med Microbiol,2013,303(6-7):396-403.
[20]	POOLE K.At the nexus of antibiotics and metals:the impact of Cu and Zn on antibiotic activity and resistance[J].Trends Microbiol,2017,25(10):820-832.
[21]	FEßLER A T,ZHAO Q,SCHOENFELDER S,et al.Complete sequence of a plasmid from a bovine methicillin-resistant Staphylococcus aureus harbouring a novel ica-like gene cluster in addition to antimicrobial and heavy metal resistance genes[J].Vet Microbiol,2017,200:95-100.
[22]	CLSI.M31-A2 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals:approved standard-2nd edition[S].Wayne,PA:Clinical and Laboratory Standards Institute,2006.
[23]	CLSI.M100 Performance standards for antimicrobial susceptibility testing[S].Wayne,PA:Clinical and Laboratory Standards Institute,2019:194-198.
[24]	吴生根.金黄色葡萄球菌对碘伏和戊二醛抗性及其机制研究[D].福州:福建医科大学,2009.WU S G.Study on the resistance of Staphylococcus aureus to Povidone Iodine、Glutaraldehyde and its mechanisms[D].Fuzhou:Fujian Medical University,2009.(in Chinese)
[25]	LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct method[J].Methods,2001,25(4):402-408.
[26]	ATSHAN S S,SHAMSUDIN M N,KARUNANIDHI A,et al.Quantitative PCR analysis of genes expressed during biofilm development of methicillin resistant Staphylococcus aureus (MRSA)[J].Infect Genet Evol,2013,18:106-112.
[27]	CHOVANOVÁ R,MIKULÁŠOVÁ M,VAVERKOVÁ Š.Modulation of mecA gene expression by essential oil from Salvia sclarea and synergism with oxacillin in methicillin resistant Staphylococcus epidermidis carrying different types of staphylococcal chromosomal cassette mec[J].Int J Microbiol,2016,2016:6475837.
[28]	HU H W,WANG J T,LI J,et al.Field-based evidence for copper contamination induced changes of antibiotic resistance in agricultural soils[J].Environ Microbiol,2016,18(11):3896-3909.
[29]	AARESTRUP F M,HASMAN H.Susceptibility of different bacterial species isolated from food animals to copper sulphate,zinc chloride and antimicrobial substances used for disinfection[J].Vet Microbiol,2004,100(1-2):83-89.
[30]	CAVACO L M,HASMAN H,AARESTRUP F M,et al.Zinc resistance of Staphylococcus aureus of animal origin is strongly associated with methicillin resistance[J].Vet Microbiol,2011,150(3-4):344-348.
[31]	黄谦,王春贵.浅谈冷季牦牛补饲的必要性[J].畜禽业,2019,30(6):29.HUANG Q,WANG C G. On the necessity of supplementary feeding of yak in cold season[J].Livestock and Poultry Industry,2019,30(6):29.(in Chinese)
[32]	HLZEL C S,MVLLER C,HARMS K S,et al.Heavy metals in liquid pig manure in light of bacterial antimicrobial resistance[J].Environ Res,2012,113:21-27.
[33]	YAZDANKHAH S,RUDI K,BERNHOFT A.Zinc and copper in animal feed-development of resistance and co-resistance to antimicrobial agents in bacteria of animal origin[J].Microb Ecol Health Dis,2014,25(1):25862.
[34]	JACOB M E,FOX J T,NAGARAJA T G,et al.Effects of feeding elevated concentrations of copper and zinc on the antimicrobial susceptibilities of fecal bacteria in feedlot cattle[J].Foodborne Pathog Dis,2010,7(6):643-648.
[35]	LECLERCQ R.Mechanisms of resistance to macrolides and lincosamides:nature of the resistance elements and their clinical implications[J].Clin Infect Dis,2002,34(4):482-492.
[36]	SMITH C A,BAKER E N.Aminoglycoside antibiotic resistance by enzymatic deactivation[J].Curr Drug Targets Infect Disord,2002,2(2):143-160.