| Antibiotics and heavy metals are widely used in animal husbandry.Antibiotics had been added to animal feed as growth promoters for a long time.However,the problem of antibacterial resistance is becoming more and more serious.In 2006,the EU banned the use of growth promoting antibiotics in animal feed.Copper has become an economic and effective alternative to antibiotics because of its advantages of promoting growth and antidiarrhea.However,in recent years,several studies have shown that copper tolerance has a synergistic effect with antibiotic resistance of some bacteria,which prompts us have to reexamine the comprehensive effect of feeding high concentration of copper on livestock production.So,is there a relationship between antibiotic resistance and copper tolerance?Does feeding high copper promote the production and maintenance of antibiotic resistance of enterococci and lactobacilli in piglets?Is there a relationship between copper tolerance and antibiotic resistance in enterococci?The purpose of this study is to provide a scientific basis for the rational application of high concentration of copper application in animal husbandry.The project is divided into the following four parts:1 Comparison of antibiotic resistance and copper tolerance of Enterococcus spp.and Lactobacillus spp.isolated from piglets and sows before and after weaningIn China,antimicrobials and copper are used extensively as growth-promoting agents in piglets.This study aimed to characterize the role of in-feed copper in the emergence of copper-tolerant and antibiotic-resistant Enterococcus and Lactobacillus isolates in Chinese pig farms.Feces of eight piglets from four litters at 7 and 55 days of age and their mothers were traced in order to isolate Enterococcus spp.and Lactobacillus spp.The minimum inhibitory concentrations of 10 antimicrobials and copper sulfate were determined using an agar dilution method.The feed levels of Cu2+ for lactating sows,suckling piglets,and weaned piglets were 6,177,and 18 mg/kg respectively.All the 136 Enterococcus isolates were sensitive to vancomycin;and the resistance rates to penicillin,enrofloxacin,and high level streptomycin increased significantly after weaning.For the 155 Lactobacillus isolates,the resistance rates to ampicillin,chloramphenicol,tetracycline,and enrofloxacin were significantly higher in weaned piglets.The ratios of copper tolerant Enterococcus and Lactobacillus isolates both increased significantly after weaning(P<0.05).A phenotypic correlation was observed after classifying the isolates into two groups(CuSO4 MIC50<16 or>=16 for enterococci;CuSO4 MIC50<12 or>=12 for lactobacilli)and comparing the antimicrobial-resistant percentage of two groups.On the species level,a significant increase of E.faecalis to enrofloxacin was associated with the increase of copper MIC(P<0.05).The findings revealed the changes of the antibiotic resistance and copper tolerance level of enterococci and lactobacilli from suckling to weaned piglets.There appears to be a strong association between in-feed copper and increased antibiotic resistance in enterococci and lactobacilli in Chinese swine farms.2 Effects of in-feed copper and sub-therapeutic enrofloxacin on fecal microbiota composition and quinolone resistance genes in Xiang pigsIn the first chapter of this dissertation,the results showed that the copper resistance of enterococci was significantly related to the enroflaxacin resistance.However,the effect of enrofloxacin on the bacterial structure and gene level was not clear.Therefore,the aim of the present study was to investigate the changes of fecal microbiota under copper and enrofloxacin pressure.Individual fecal samples were collected from Xiang pigs in the control and copper groups on days 14,19,24,34,and 48.Both groups were given a sub-therapeutic dose of enrofloxacin in feed from days 15 to 24.PCR-DGGE and Miseq high-throughput sequencing were used to investigate the effects.PCR-DGGE showed that the fecal microflora on days 14 and 19 could be clustered into one cluster according to the trial period and diet treatment;the fecal microflora on day 14 and 19 days,and day 24 and 48 could be clustered into one large cluster separately.The high throughput sequencing showed that Firmicutes,Bacteroidetes,and Proteobacteria were the dominant phylum in two groups.Enrofloxacin only had a significant influencing on Proteobacteria.Both of copper an enrofloxacin decreased the abundance of genus Pseudobutyrivibrio and Faecalibacterium.The abundance of OTU1094(g:Prevotella)in copper group was significantly lower than in control group.However,the abundance of Campylobacter in copper group was significantly higher(P=0.030)after enrofloxacin treatment.The principle reactive analysis on a genus level revealed that enrofloxacin strongly diminished the differences caused by copper between the two groups.A correlation analysis showed that copper was positively correlated with the phylum Spirochaetae,genus Bacteroides and Ruminococcus,and negatively correlated with Erysipelotrichaceae_unclassified,OTU1094(g:Prevotella),and OTU779(g:Solobacterium).No significant change was observed in genes tetM,ermB,and aac(6’)-Ib-cr.The results revealed that both copper and enrofloxacin could change the microbiota of Xiang pigs significantly,but enrofloxacin alone had quite limited influence at the phylum level.Otherwise,even a sub-therapeutic dose of enrofloxacin could have a stronger effect than copper.High copper in feed could reduce the relative abundance of some opportunistic pathogens.However,under the pressure of both copper and enrofloxacin,the abundance of Campylobacter could increase,suggesting that we should reevaluate the potential risk of copper and antibiotics used as feed additives.3 Effects of in-feed copper and sub-therapeutic enrofloxacin on antibiotic resistance of Enterococcus spp.and Lactobacillus spp.in feces from weaned Xiang-pigsThe experiment was aimed to study the effect and association of in feed-copper and enrofloxacin on antibiotic resistance and copper tolerance of Enterococcus spp.and Lactobacillus spp.in feces from Xiang-pigs,in order to supply some basic information to animal husbandry.Individual fecal samples were collected from Xiang pigs in the low(the copper concentration is 6 mg/kg)and high copper(125 mg/kg)groups on days 14,19,24,34,and 48.Both groups were given a sub-therapeutic dose of enrofloxacin in feed from days 15 to 24.Enterococci and lactobacilli were isolated from fecal samples of which enrofloxacin and copper susceptibilities were dectected using the agar dilution method recommended by CLSI.The results showed that in high copper group,the enrofloxacin resistance rate and copper tolerance rate of enterococci increased(P<0.001)on day 24 and decreased(P<0.001)on day 34 significantly;in low copper group,few changes were observed.For lactobacilli,the copper tolerance in high copper group increased significantly(P<0.001)on day 24 and kept increasing on day 34;in low copper group,the increase appeared on day 34 and it decreased significantly on day 48(P<0.001).The changes in enrofloxacin resistance in lactobacilli were similar to those in copper tolerance.Furthermore,the copper tolerance of enterococci in high copper group was significantly higher than that in low copper group.The enrofloxacin resistance and copper tolerance of lactobacilli in high copper group on day 24 were both significantly higher than in low copper group.No changes were observed on day 48 for both resistance.We also found the association between enrofloxacin resistance and copper tolerance.In conclution,in-feed copper and enrofloxacin have effects on the antibiotic resistance and copper tolerance of enterococci and lactobacilli.High-level copper may facilitate and maintain the antibiotic resistance and copper tolerance in enterococci and lactobacilli.4 Antibiotic resistance and copper tolerance of E.faecium and the induction of E.faecium by copper and enrofloxacin in an in vitro modelThe results of the third chapter show that,on the genus level,copper and sub-therapeutic enrofloxacin could increase the antibiotic resistance and copper resistance of Enterococcus,Copper promotes the production of enrofloxacin resistance of Enterococcus spp.However,it is still unknown how the enrofloxacin resistance and copper tolerance will change on species level under antibiotic and copper pressure.The purpose of this study was to explore the susceptibility of different antimicrobial agents and the prevalence of resistance genes in Enterococcus faecium,and on this basis,to investigate the relationship between different resistance genes and different antimicrobial phenotypes in vitro by induction test.One hundred and thirty-eight strains of Enterococcus faecium isolated from the study described in chapter three were identified by multiplex-PCR.The strains were analyzed by pulsed field gel electrophoresis and the sensitivity and resistance genes were detected by agar dilution and PCR.The effects of different doses of antimicrobial agents on the sensitivity of Efaecium were also investigated.The results showed that 138 strains of Efaecium were divided into 55 different PFGE genotypes.PFGE genotype 2 was found in day 0,day 19 and day 24 in both groups.The positive rate of tcrB was 60%(33/55),and that of cueO was 65.5%(36/55).Among these isolates,32 strains of E.faecium carried tcrB and cueO simultaneously.The correlation analysis showed that the tolerance level of tcrB/cueO positive isolates to penicillin(P=0.000),ampicillin(P=0.000),florfenicol(P=0.000),erythromycin(P=0.000),gatifloxacin(P=0.041)and copper(P=0.000)was significantly higher than that of tcrB/cueO negative isolates.The MDR level of tcrB/cueO positive isolates was also significantly higher than that of tcrB/cueO negative isolates(P=0.012).The gene tcrB was positively correlated with cueO(P=0.000)and ermB(P=0.003).The induction test showed that the sensitivity to quinolones decreased in different degrees under the long-term pressure of low-dose enrofloxacin and copper sulfate,and the MIC value increased by 2-4 times,but the sensitivity to non-quinolones and copper remained unchanged.In conclusion,copper resistance is related to the resistance to penicillins,amidohols and macrolides.There is the possibility of horizontal transmission of resistance genes at the same time.In vitro,the pressure of antibiotics has a selective effect on antimicrobial resistance.In conclusion,the results of this study can be concluded as follows:1.In pig farm,the antibiotic resistance of enterococci and lactobacilli are serious especially to tetracyclines and macrolides.The resistance of enterococci and lactobacilli to enrofloxacin and copper in weaned piglets was significantly higher than that in suckling piglets.There is a correlation between copper tolerance and enfloxacin resistance in enterococci and lactobacilli.2.Feeding high copper(125 mg/kg)and sub-therapeutic enrofloxacin can significantly change the composition of pig fecal microbiota.The sub-therapeutic enrofloxacin can have a stronger effect on fecal microbiota than copper.Enrofloxacin and copper can reduce the relative abundance of some conditional pathogens,but also the relative abundance of bacteria which can produce butyric acid.3.The high copper and sub-therapeutic enrofloxacin can significantly increase and maintain the antibiotic resistance and copper tolerance of enterococci and lactobacilli.There is a correlation between copper tolerance and enrofloxacin resistance,especially in enterococci.4 tcrB and cueO genes are the main mechanisms of copper tolerance in Enterococcus faecium.tcrB/cueO was positively correlated with phenotype resistance of penicillin,erythromycin,florfenicol and erythromycin;tcrB was positively correlated with cueO and ermB.In vitro,low dose of enrofloxacin and low dose of copper can induce the decrease of the susceptibility of Enterococcus faecium to fluoroquinolones. |
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