饲喂不同抗生素对肉牛废弃粪便中四环素耐药基因数量及持久性的影响

通过对肉牛饲喂低于治疗水平的不同种类抗生素,研究其对肉牛废弃粪便微生物群落中四环素耐药基因数量与持久性的影响。试验肉牛被分成不同抗生素处理组,即氯四环素组、氯四环素和磺胺甲嘧啶组及对照组。将每个围栏中所有动物的新鲜粪便混匀为一份混合样品作为模式样品(每个处理组3份),分别露天放置,在第7、14、28、42、56、70、84、98、112、126和175 天时采样并提取DNA,利用Real-time PCR方法测定四环素耐药基因tet(B),tet(C),tet(L),tet(M),tet(W)及16S rRNA的浓度。结果显示,16S rRNA的浓度在不同处理间相似,在56 d内均有增加的趋势(P＜0.05);总体上看,四环素组的耐药基因初始浓度较高(P＜0.05);所有处理组的tet(B)和tet(C)浓度到56 d时均增长了1~2个对数级,到175 d时又降低到初始水平,而tet(M)与tet(W)的浓度与其他耐药基因相比较高。因此,四环素耐药基因可以在废弃粪便中持续存在超过175 d,而某些基因的最初数量可能会导致错估其后的变化,其浓度的暂时改变并不能归因于微生物群落数量的变化。     

广东地区水禽源大肠杆菌对四环素的耐药性及耐药基因检测

采用琼脂二倍稀释法测定256株水禽大肠杆菌分离株对四环素类药物的敏感性,并通过PCR方法调查分离株携带耐药基因tet A、tet B、tet C和tet M的情况。药敏试验结果表明256株水禽大肠杆菌分离株对四环素和多西环素耐药率分别为92.2%和77.3%。在237株大肠杆菌四环素耐药株中,tet A、tet B、tet C和tet M的携带率分别为49.8%、57.8%、49.4%和40.1%,仅8.0%的耐药株没有检测到4种耐药基因。结果表明:tet A、tet B、tet C和tet M广泛存在于水禽源大肠杆菌中。tet A和tet B对水禽源大肠杆菌四环素耐药株的产生起重要作用,主动外排作用是大肠杆菌对四环素产生耐药性的主要机制。     

青岛地区鸭源四环素类耐药大肠杆菌流行情况调查及耐药基因分析

【目的】通过对山东青岛地区鸭源样品进行四环素类耐药大肠杆菌的分离鉴定,了解其流行分布规律。对耐药菌株进行药敏检测及耐药基因鉴定,研究四环素类耐药大肠杆菌多重耐药率及耐药基因携带情况,为抗生素的合理应用提供理论依据。【方法】以肉鸭泄殖腔拭子及盲肠样本为试验材料,通过四环素选择培养基分离大肠杆菌。采用琼脂稀释法测定耐药菌株对12种抗菌药物的敏感性,通过PCR检测四环素类耐药大肠杆菌携带耐药基因情况,进一步通过接合试验评估tet(X)基因的可转移性,进而对部分tet(X)基因阳性菌株进行全基因组测序,同时探究tet(X)耐药基因的遗传环境特征。【结果】采集的63份样品经分离鉴定,获得48株四环素耐药大肠杆菌,分离率为76.19%。药敏试验结果显示,所有菌株对四环素和土霉素耐药,对多西环素和氟苯尼考耐药率分别为93.75%和89.59%,且对多黏菌素、替加环素及美罗培南耐药率分别为64.58%、16.67%和4.17%;所有菌株均存在多重耐药现象,其中以耐7种药物最多。耐药基因检测结果与耐药情况基本相对应,耐药率高的药物相应耐药基因呈现较高携带率;其中floR、tet(A)、qnrS、mcr-...     

247株猪源沙门菌对四环素类药物耐药性和耐药基因的检测分析

为探究猪源沙门菌分离株对四环素类抗菌药物耐药性及四环素耐药基因(tet)的流行与分布情况,从7省的合作猪场采集病死猪肝脏、肺脏、肠道作细菌分离,利用沙门菌属特异性侵袭基因(invA)和16S rDNA扩增、测序等方法进行鉴定,并用微量肉汤稀释法测定沙门菌分离株对四环素类抗菌药物的敏感性,PCR方法测定tet基因,以及ERIC-PCR方法分析猪源沙门菌之间的相关性和遗传关系。从823份病料中分离鉴定出247株猪源沙门菌,分离率为30.01%。药敏结果显示,对多西环素、土霉素的耐药率分别为87.45%和94.74%。PCR检测发现,分别有62,66,3株单独携带tetA、tetB、tetC基因;分别有38,8,4,7,1株菌同时携带两种不同的tet基因(tetA+B、tetA+C、tetA+D、tetB+C、tetC+D);分别有9,6,1,6,1,4,3株菌同时携带3种不同的tet基因(tetA+B+C、tetA+B+D、tetA+B+M、tetA+C+D、tetA+D+M、tetB+C+D、tetB+C+M);分别有6,2株菌同时携带4种不同的tet基因(tetA+B+C+D、tetA+C+D+M);没有检测到单独携带tetD、tetM以及同时携带5种tet基因的菌株。接合试验表明,tetM可与tetA或tetC共同在沙门菌与大肠杆菌间转移,导致菌株多西环素抗性水平转移。试验菌共分为A~δ共30个ERIC型,tet基因分布于不同ERIC型菌株中,表明其在试验菌株间水平扩散。本试验首次在猪源沙门菌中发现了编码核糖体保护蛋白的四环素类耐药基因tetM。猪源沙门菌对四环素类药物耐药严重,对多西环素、土霉素具有普遍耐药性,tetA、tetB基因在7省猪源沙门菌中流行最广泛,分离菌对四环素类药物的普遍耐药性与单个或多个tet基因的普遍存在密切相关。     

牛肉源大肠杆菌的耐药性检测及相关耐药基因分布

为了解牛肉源大肠杆菌的耐药性并检测其相关耐药基因分布,本研究选取了117株牛肉源大肠杆菌,经药敏纸片法对11种抗菌药物进行了药敏检测,并根据耐药表型利用普通和(或)多重PCR技术对耐四环素菌中tet(A)、tet(B)和tet(C)基因,耐氨苄西林菌中blaTEM1、blaPSE1和blaOXA1基因,耐链霉素菌中strA-strB、aadA1基因,耐磺胺类药菌中sul1、sul2和sul3基因进行了调查分析。结果显示,117株大肠杆菌对四环素、氨苄西林、链霉素和磺胺异恶唑的耐药率较高,分别为89%、42%、38%和22%。tet(A)基因是所有四环素耐药基因中最为流行的一种基因(55%);在检测的3个β-内酰胺类药物耐药基因中,最流行的为blaTEM1基因(73%);链霉素的耐药性主要由strA-strB基因(38%)编码;sul2基因在耐磺胺异恶唑菌中的检出率最高(77%)。结果表明本次分离的牛肉源大肠杆菌耐药非常严重,进一步肯定了肉牛业生产中抗菌药的使用对大肠杆菌耐药性的产生和发展所发挥的重要作用,提示食品动物养殖应严格控制饲料中抗菌药的滥用。     

禽源沙门菌对四环素的耐药性及耐药基因调查分析

为了更好地了解禽源沙门菌对四环素的耐药性及耐药基因分布,从不同来源的家禽样品中分离沙门菌,调查其对四环素的耐药性以及耐药菌株中8种四环素耐药基因[tet(A)、tet(B)、tet(C)、tet(W)、tet(M)、tet(D)、tet(K)和tet(L)]的携带情况.结果表明,18.8%的沙门菌分离株对四环素耐药,健康成鸡分离株对四环素的耐药性明显高于雏鸡、死胚或病禽分离株;四环素耐药株中tet(A)、tet(B)和tet(M)基因的携带比例分别为73.1%、11.5%和3.8%,说明沙门菌对四环素的耐药机制以tet(A)和tet(B)基因介导的主动外排为主.本研究首次在对四环素耐药的沙门菌中检测到tet(M)基因,说明tet(M)基因在沙门菌对四环素的耐药性方面也具有潜在的作用.     

一株致猪肺炎大肠杆菌的分离鉴定及其耐药表型、耐药基因型分析

为了了解猪肠外致病性大肠杆菌的耐药性,试验采用菌落形态观察、小鼠毒力试验、回归动物试验和PCR等方法对1株引起猪肺炎的病原菌进行鉴定,并通过微量稀释法进行耐药性检测,在全基因组测序的基础上对耐药基因型进行分析。结果表明:该病原菌是肠外致病性大肠杆菌,呈多重耐药,其染色体基因组携带有外排调控基因Mar C、SoxR,外排泵基因MATE和ABC,耐药基因移动原件IS,四环素耐药基因tet,氟苯尼考耐药基因FloR及氯霉素乙酰转移酶基因等。     

广东省某大型养鸡场tet(X4)阳性大肠杆菌流行分布特征

2020年8月从广东省某大型养鸡场不同区域采集粪便、土壤、水体等样品共654份，进行替加环素耐药大肠杆菌的分离鉴定；PCR方法检测替加环素耐药大肠杆菌的tet(X4)基因；PFGE分型检测tet(X4)阳性大肠杆菌的克隆传播情况；采用琼脂二倍稀释法和肉汤二倍稀释法测定tet(X4)阳性大肠杆菌的最小抑菌浓度(MIC);接合转移试验检测tet(X4)水平传播情况。结果显示，共分离得到204株替加环素耐药大肠杆菌，检测出165株tet(X4)阳性大肠杆菌，检出率为25.23%(165/654)。根据菌株来源和分离率，挑选72株进一步研究tet(X4)阳性大肠杆菌在该养鸡场的流行特征。PFGE分型将72株tet(X4)阳性大肠杆菌分为39个簇，表明该养鸡场无明显克隆传播现象。tet(X4)阳性大肠杆菌对替加环素的MIC范围为8～16 mg/L,且表现多重耐药表型，对四环素、多西环素、米诺环素、氨苄西林、氟苯尼考均表现高水平耐药，而对黏菌素、美罗培南均敏感。接合转移试验表明，tet(X4)阳性大肠杆菌主要位于IncHI1型质粒上(93.06%)。结果表明，tet(X4)基因在该养殖场主要随In...     

鸭源致病性大肠杆菌四环素类耐药基因检测

对40株鸭源致病性大肠杆菌的四环素类耐药基因进行检测并分析,利用PCR技术扩增主动外排机制耐四环素类抗生素的耐药基因tet A、tet B、tet C、tet D、tet K、tet L。结果表明,tet B、tet A、tet C、tet D的检出率分别为80.0%、75.0%、55.0%、15.0%,tet K和tet L的检出率均为0%,tet B基因的检出率最高。     

四环素耐药基因在鸡源沙门氏菌中的分布和传播

试验分析了84株鸡源沙门氏菌分离株的四环素耐药性,用PCR方法检测四环素耐药基因在分离株中的分布情况。结果显示,四环素耐药率为49%(41/84),鸡白痢和鸡伤寒沙门氏菌仅携带tet(A)基因(23/23),肠炎沙门氏菌和德尔卑沙门氏菌携带tet(A)(8/18)、tet(B)(17/18)或tet(G)(10/18)三种基因,tetC基因在这些沙门氏菌中都没有检测到。该类基因多数位于结合性质粒上,但是不在整合子范围内。     

Susceptibility of Escherichia coli and Enterococcus faecium isolated from pigs and broiler chickens to tetracycline degradation products and distribution of tetracycline resistance determinants in E. coli from food animals

One hundred Escherichia coli isolates from diseased and healthy pigs, cattle and broiler chickens were screened for the presence of tetracycline resistance genes tet(A), (B), (C), (D) or (E). The tet(A) gene was the most abundant (71% of the 100 isolates) followed by tet(B) (25%). The predominance of tet(A) and tet(B) applied to all three animal species, and there was no difference between the distribution of tet(A) and tet(B) genes among non-pathogenic and pathogenic E. coli in any of the animal species. The susceptibility of 20 of these isolates together with 10 tetracycline sensitive E. coli and 18 tetracycline resistant and 10 sensitive Enterococcus faecium to tetracyclines and tetracycline degradation products was determined. The resistant isolates showed reduced resistance to anhydrotetracycline, 4-epi-anhydrotetracycline, anhydrochlortetracycline and 4-epi-anhydrochlortetracycline. In general both the tetracycline resistant and susceptible E. faecium were more susceptible to the compounds tested than E. coli.     

Genetic diversity and tetracycline resistance genes of Histophilus somni

Nasal swabs were collected at three time points from 2378 calves in four feedlots and cultured for Histophilus somni to assess genetic relatedness and tetracycline resistance. The proportions of animals carrying tetracycline resistant isolates were 0.32% at arrival, 14.82% at interim, and 0.80% at exit. The 606 H. somni isolates recovered were compared by pulsed-field gel electrophoresis (PFGE), screened for the presence of plasmids, and assessed for the tetracycline resistance genes tet(A), tet(B), tet(C), tet(E), tet(G), tet(H), tet(K), tet(L), tet(M) and tet(O) using multiplex polymerase chain reaction. Most of the isolates (98.6%) belonged to one of seven PFGE clusters (A-G) of closely related profiles with 77.7% of the isolates belonging to clusters C and D. Clusters A, B and E were associated with a higher proportion of tetracycline susceptible isolates. Genetic diversity of the isolates was highest at entry in the feedlot and lowest after the period when the animals received in-feed chlortetracycline (interim samples). Clusters A and E were more prominently represented at exit from the feedlot than other clusters. All resistant strains harboured the gene tet(H) while no other tetracycline resistance genes and no plasmids were detected with the methodology employed. It appears that genetic variability in H. somni in Alberta feedlots is low, dissemination likely occurs by clonal expansion, and resistance to tetracyclines is mediated by the tet(H) encoded efflux pump. Pulsotypes associated with tetracycline susceptible strains appear more common at exit suggesting that the in-feed oxytetracycline included throughout the feeding period is not sufficient to exert selective pressure for resistant strains.     

贵州省猪源大肠杆菌对四环素类药物的耐药性及耐药基因检测

为考察贵州省猪源大肠杆菌对四环素类抗菌药的耐药情况和耐药基因的流行情况,本试验采用微量肉汤稀释法测定783株分离于贵州省8个地区规模化养殖场的猪源大肠杆菌对6种四环素类药物的敏感性,并通过PCR方法检测其携带四环素耐药基因情况。结果显示,猪源大肠杆菌对土霉素、四环素、金霉素、多西环素、米诺环素和替加环素的耐药率分别为97.3%、97.6%、96.6%、89.3%、48.0%和34.2%;土霉素和四环素的耐药水平最高,其MIC₅₀分别为256 和128 μg/mL,而MIC₉₀均为512 μg/mL,四环素的耐药倍数高于土霉素;米诺环素和替加环素的耐药水平最低,其MIC₉₀分别为32和4 μg/mL,耐药倍数相同;耐药基因tetA、tetB、tetC、tetD和tetM的检出率分别为92.60%、41.50%、58.75%、58.62%和70.10%;耐药菌株中主要以tetA基因为主,且大多数以携带复合基因的形式存在;大肠杆菌对土霉素、四环素、金霉素、多西环素和米诺环素的耐药性分别与耐药基因tetB和tetM呈极显著相关（P<0.01）,tetD基因分别与对米诺环素和替加环素的耐药性呈极显著相关（P<0.01）。贵州省猪源大肠杆菌对四环素类药物的耐药情况十分普遍,尤其是四环素和土霉素,金霉素和强力霉素有高度耐药的趋势;外排泵是四环素类药物主要的耐药机制;在兽医临床上越来越严重的耐药情况与耐药基因的广泛存在有很大关系。     

Identification of the tetracycline resistance gene, tet(M), in Erysipelothrix rhusiopathiae

This is the first report to demonstrate the presence of tet(M) in naturally occurring isolates of tetracycline-resistant Erysipelothrix rbusiopathiae, which causes swine erysipelas. The tet(M) gene was isolated from E. rhusiopathiae strain KY5-42. The nucleotide and the deduced amino acid sequence were 99% identical to the tet(M) gene from Enterococcus faecalis. The gene was necessary and sufficient for the expression of tetracycline resistance in Escherichia coli. The presence of the tet(M) gene in the 114 tetracycline-resistant E. rhusiopathiae isolates from diseased pigs was detected by the polymerase chain reaction assay. The specific amplified DNA fragment was obtained from all 114 tetracycline-resistant strains. It was suggested that the tet(M) gene was widely present in the field isolates of E. rhusiopathiae resistant to tetracycline.     

Antimicrobial susceptibility of enteric bacteria recovered from feedlot cattle administered chlortetracycline in feed

OBJECTIVE: To evaluate administration of chlortetracycline in feed of cattle as a method to select for tetracycline resistance among enteric bacteria in feedlot settings. ANIMALS: 20 steers. PROCEDURES: Steers were randomly assigned to an exposed cohort (n = 10) or an unexposed cohort (control cohort; 10). Chlortetracycline (22 mg/kg) in cottonseed meal was administered to the exposed cohort on days 0 through 4, 6 through 10, and 12 through 16. The control cohort was administered only cottonseed meal. Fecal samples were collected from 16 steers on days -7, 0, 2, 6, 8, 12, 14, 19, 22, 26, and 33, and Escherichia coli and Enterococcus spp were isolated. Minimum inhibitory concentration (MIC) of selected antimicrobials was estimated. RESULTS: Overall, 56.0% and 31.4% of E coli and Enterococcus isolates, respectively, were resistant to tetracycline. Exposure to chlortetracycline was associated with a significant temporary increase in log(2) MIC for both genera but returned to preexposure values by day 33. Averaged across time, the proportion of tetracycline-resistant E coli and Enterococcus isolates was significantly greater in exposed than in unexposed steers. Although all ceftiofur-resistant E coli isolates were coresistant to tetracycline, exposure to chlortetracycline led to a significant decrease in the proportion of E coli resistant to ceftiofur during exposure. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to chlortetracycline was associated with a temporary increase in the likelihood of recovering resistant bacteria. Exposure to chlortetracycline decreased the likelihood of recovering ceftiofur-resistant E coli isolates, even though isolates were coresistant to tetracycline. These findings warrant further investigation.     

Associations between antimicrobial use and the prevalence of antimicrobial resistance in fecal Escherichia coli from feedlot cattle in western Canada

A randomized, controlled, blinded clinical trial was performed at a research feedlot in western Canada. Auction-market-derived steers (n = 288) were randomly assigned to 1 of 3 treatments: 1) no antimicrobials on arrival; 2) oxytetracycline in the starter ration for 14 d; and 3) long-acting oxytetracycline subcutaneously on day 0. Minimal inhibitory concentrations of 7 antimicrobials were determined for 3 generic fecal E. coli isolates per animal on arrival and throughout the feeding period. There was a low prevalence of antimicrobial resistance in generic E. coli isolates from calves on arrival. There were increased proportions of cattle with resistant E. coli isolates early in the feeding period among calves in groups 2 and 3. Individual animal treatments were not associated with increased proportions of cattle with resistant E. coli isolates preslaughter. There was no difference in the proportion of animals with E. coli isolates resistant to tetracycline between the treatment groups preslaughter. However, there were significantly more animals with tetracycline resistant isolates of E. coli preslaughter than at arrival.