沙门菌体内耐药性诱导模型的建立和体内外诱导耐药菌基因差异分析

以秀丽隐杆线虫N2野生型为宿主,将鼠伤寒沙门菌ATCC13311定植于线虫体内,然后使用梯度浓度递增法提高培养液中的环丙沙星浓度,诱导沙门菌在线虫体内产生耐药性,同时进行体外诱导耐药试验。对体内、外诱导耐药菌的gyrA、gyrB、parC和parE基因的氟喹诺酮耐药决定区(Quinolone resistance determining regions,QRDR)和外排泵抑制子基因(acrR、marR、ramR和soxR)进行PCR扩增、测序和分析。结果表明,鼠伤寒沙门菌ATCC13311定植于线虫体内,经过诱导后得到环丙沙星MIC为4 mg/L的耐药菌TN4,体外诱导试验获得环丙沙星MIC为4μg/mL的耐药菌TW4。TN4的gyrA基因发生了Asp87Asn突变。体外诱导耐药菌TW4的gyrA基因发生了Ser83Phe和Asp87Val突变,ramR基因出现了20bp的缺失。本研究建立了鼠伤寒沙门菌-秀丽隐杆线虫体内耐药性诱导模型,并比较了体内、外诱导耐药菌的基因突变差异,为进一步研究细菌在动物体内的耐药机理奠定了基础。     

喹诺酮类耐药决定区、外排泵负调控基因对大肠杆菌氟喹诺酮高水平耐药分子机制的影响

为了探讨耐喹诺酮类决定区(QRDR)、外排泵负调控基因(acrR、marR和soxR)突变对临床分离株氟喹诺酮(FQs)高水平耐药的影响,本研究对临床分离的18株FQs耐药大肠杆菌(E.coli),采用PCR方法检测QRDR、acrR、marR和soxR的突变情况;通过RT-PCR的方法检测外排泵及膜孔蛋白相关基因的表达水平。结果显示,QRDR的突变主要集中在常规突变GyrA(Ser83Leu 和 Asp87Asn)和ParC(Ser80Ile),同时也检测出稀有突变ParC Glu84Gly、Glu84Lys、Glu84Val和Glu84Ala,ParE Ser458Ala等。ED28在acrR基因存在777 bp插入序列;12株菌(包括ATCC25922)在MarR存在Gly103Ser和Tyr137His双突变,其中EP26和EG42存在插入片段;ED40在SoxR存在Thr38Ser、Gly74Arg氨基酸替换。在多突变药菌株中,AcrAB的表达水平明显升高,OmpC和OmpF表达量降低、甚至缺失。     

抗生素压力下不同血清型沙门氏菌耐药性研究

本研究旨在探讨不同血清型沙门氏菌在环丙沙星抗生素压力下突变频率及在耐药发展过程中靶位基因突变、外排泵及调控基因表达的差异。选取临床分离的印第安纳型、肠炎型和鼠伤寒型沙门氏菌的敏感菌株,在环丙沙星压力下诱导耐药突变,分别获得一系列不同程度的耐药突变株。分别检测不同血清型沙门氏菌突变株的突变频率、靶位基因喹诺酮耐药决定区(QRDRs)和外排泵调控基因ramR-ramA突变及外排泵相关基因的表达水平;同时检测了母株在羰基氰化物间氯苯腙(CCCP)存在情况下环丙沙星药物的蓄积浓度,以确定母株是否存在外排泵的作用。结果表明,在环丙沙星压力下,印第安纳型沙门氏菌较肠炎型和鼠伤寒型有更高的突变频率,易获得耐药株;印第安纳血清型菌株耐药性的获得主要是由于靶位基因gyrA发生单突变,协同外排泵外排作用增强而获得高水平耐药;肠炎型沙门氏菌耐药性获得主要是由于靶位基因gyrA发生83和87位双位点突变,并随着gyrB和parC基因的多位点同时突变而获得高水平耐药,耐药性的发展过程中没有外排泵作用参与;而鼠伤寒沙门氏菌在抗生素压力下不易发展成耐药菌,耐药性发生主要是由于靶位基因gyrB发生突变,而伴随parC基因突变及微弱的外排泵作用导致耐药水平增加。     

MarA和SoxS共同调节大肠杆菌K12外排泵AcrAB-TolC的表达

利用Red同源重组技术构建大肠杆菌K12调控基因缺失菌株,探讨耐药发展过程中marRAB和soxRS对大肠杆菌K12外排泵AcrAB-TolC表达的调控作用。在环丙沙星浓度递增的条件下诱导K12及调控基因缺失菌株,分别获得系列突变菌株,测定各菌株对其他药物的MIC,并检测靶位基因突变情况。利用RT-PCR方法检测诱导突变株中外排泵基因、调控基因、膜孔蛋白基因的表达水平。结果表明,K12正向调控基因(marA和soxS)缺失株在环丙沙星选择压力下仅获得环丙沙星敏感性降低的菌株,而负向调控基因(marR和soxR)在环丙沙星选择压力下可获得环丙沙星中介和耐药的菌株,且诱导突变株仅出现与耐药有关的gyrA单突变(Ser83Leu和Asp87His)。正向调控基因marA和soxS缺失后,外排泵基因表达水平变化不明显,而负向调控基因marR和soxR缺失后,marA和soxS的表达水平显著升高,引起外排泵基因表达水平显著升高,最高达到约13倍。当阻遏蛋白SoxR被敲除后,soxS的表达水平没有变化,而marA的表达水平显著升高,外排泵基因acrB表达水平也升高,说明在调控AcrAB-TolC外排泵基因表达方面,marRA与soxRS存在功能上的冗余,即当其中一个调控因子功能缺陷时,另一个调控因子可以发挥互补调控功能。因此,MarA和SoxS对三聚体系统AcrAB-TolC的正向调控作用是通过解除负向调控蛋白MarR和SoxR对其的阻遏作用实现的,MarRA和SoxRS共同调节外排泵AcrAB-TolC的表达水平。     

广州市生鲜鸡中肠炎沙门菌的监测及耐药基因的分子鉴定

本研究旨在了解广州市鸡肉中沙门菌的流行情况、血清型分布及肠炎沙门菌耐药性与耐药基因的携带情况。对采集自广州市部分农贸市场的鸡肉样品,参照国标检测方法(GB4789.4-2016)进行分离培养并使用泰国SA公司沙门菌属诊断血清进行血清分型;用Kirby-Bauer法测定肠炎沙门菌对16种抗菌药物的耐药性,用PCR方法检测肠炎沙门菌携带的耐药基因。结果显示:316份鸡肉样品中,阳性率为76.9%。共鉴定23种血清型,主要血清型为阿贡纳(Salmonella Agona,19.8%)、科瓦利斯(S.Kottbus,14.0%)、姆班达卡(S.Mbandaka,11.9%)、肯塔基(S.Kentucky,10.3%)、肠炎(S.enteritidis,7.8%)、布伦登卢普(S.Braenderup,7.4%)。耐药性结果显示:肠炎沙门菌对萘啶酸耐药率最高(100.0%),其次是磺胺复合物(79.0%)、氨苄西林(57.9%)、链霉素(36.8%)、四环素(21.0%)、庆大霉素(21.0%)、头孢噻肟(15.8%)、头孢他啶(5.3%),多重耐药率为68.4%。肠炎沙门菌中喹诺酮耐药决定区基因gyrA、gyrB、parC的突变率分别为94.7%、73.7%、15.8%,最常检出突变为苯丙氨酸-414→丝氨酸(73.7%),其次是天冬氨酸-87→甘氨酸(42.1%)、天冬氨酸-87→色氨酸(31.6%)、丝氨酸-83→色氨酸(21.1%);喹诺酮类耐药质粒的检出率较低。β-内酰胺类耐药基因blaTEM、blaCTX-M的检出率分别为42.1%、10.5%。结果显示,广州市鸡肉中沙门菌的污染率较高,血清型复杂。肠炎沙门菌对萘啶酸、磺胺甲基异恶唑、氨苄西林等常用抗生素的耐药情况较为严重,多重耐药率高。肠炎沙门菌的喹诺酮耐药决定区基因突变率高,与肠炎沙门菌对喹诺酮类药物的耐药结果具有高度的一致性。     

徐州及周边地区鸭源沙门菌的分离鉴定及其耐药性、毒力基因分析

旨在了解徐州及周边地区鸭源沙门菌血清分布、耐药状况及毒力基因携带情况。采集徐州及周边地区病鸭脏器329份进行沙门菌分离、双重PCR鉴定及血清分型;采用Kirby-Bauer(K-B)法进行抗菌药物敏感性试验;设计10对引物,对6大类耐药基因进行检测;PCR检测与沙门菌致病性相关的8个毒力基因。结果:共分离49株鸭源沙门菌,分离率为14.9%;鉴定出13种血清型,优势血清型为印第安纳沙门菌、阿贡纳沙门菌和鸭沙门菌。耐药性结果显示:分离株对氨苄青霉素(AMP)和头孢曲松(CRO)耐药率为100%,对土霉素(OXY)、阿莫西林/克拉维酸(AMC)、卡那霉素(KAN)、利高霉素(NA)、强力霉素(DOX)、恩诺沙星(ENR)、环丙沙星(OFL)抗菌药物的耐药率均超过50%以上。耐药基因blaTEM-1检出率为100%,blaPSE-1、aadA1、aadA2、tetA、gyrA、sul2基因检出率超过50%以上,parC、floR和clmA基因检出率分别为44.8%、30.6%和26.5%。毒力基因检测发现spvR(12.2%)携带率较低,mgtC和stn基因检出率为100%,invJ、sseL、siiE、sopB以及fimA基因携带率分别为75.6%、53.3%、89.6%、98.2%和97.9%。研究结果表明,徐州及周边地区鸭沙门菌血清型分布较广,分离株的耐药性及多重耐药较严重,耐药基因和毒力基因分布广泛。     

山东省肉鸡屠宰生产链中沙门菌耐药性分析及毒力基因检测

通过调查山东省不同市肉鸡屠宰生产链中沙门菌分离株血清型、耐药性及毒力相关基因,为肉鸡的健康安全生产提供数据支撑。对前期获得的233株沙门菌通过血清型快速分型试剂盒鉴定其血清型,采用最小抑菌浓度(MIC)进行13种抗菌药物的药敏试验;应用PCR技术检测位于菌株毒力岛(SPI)和毒力质粒(spv)上共15种毒力基因的携带情况。结果显示,233株沙门菌共分属25种不同的血清型,其中肠炎沙门菌、印第安纳沙门菌、汤普逊沙门菌和德尔卑沙门菌为优势血清型。233株肉鸡屠宰生产链中沙门茵对庆大霉素的耐药率最高(100%);对多西环素、氨苄西林、大观霉素、四环素、氟苯尼考和磺胺异恶唑耐药率分别为84.94%、75.73%、67.78%、56%、52%和50%。72%的菌株表现为多重耐药(≥3),产生了57种耐药谱型。沙门菌的毒力岛基因sptP、sseL、mgtC、siiE、sopB的携带率是100%,毒力基因spvA、spvB、spvC、spvD、spvR、iacP、avrA、prgK、fima和stn的检出率分别是60%、34%、49%、51%、63%、99%、38%、99%、99%和99%。结果表明,在4种优势血清型中并非均出现高耐药性,出现高耐药性的血清型主要以汤普逊沙门菌为主,汤普逊沙门菌相对于其他血清型毒力基因的携带率较高,沙门菌耐药性和毒力基因的携带间并没有明显的关联性。     

猪源链球菌对环丙沙星耐药性与耐药基因突变的相关性研究

通过微量稀释法测定28株猪源链球菌对环丙沙星的MIC值,研究东北地区猪源链球菌对环丙沙星耐药性与parC、gyrA基因突变的相关性.通过PCR方法扩增parC和gyrA基因喹诺酮耐药决定区(QRDR)并测序分析;18株耐药菌在parC基因80位的突变(AGC→ATT)导致氨基酸Ser→Ile突变,11株高度耐药菌在gyrA基因81位的突变(CAG→)CAT、CTT或CTA)导致氨基酸Ser→Ile、Phe或Tyr的突变.当菌株对环丙沙星的MIC值≤1μ/mL时,parC和gyrA基因的QRDR区均未有突变;而当MIC ≥2μg/mL时,ParC的氨基酸发生了Ser80→Ile的突变,同时发生GyrA氨基酸Ser81突变的菌株,耐药水平很高.研究表明,环丙沙星低水平类耐药是由于拓扑异构酶Ⅳ改变引起,而高水平耐药是由拓扑异构酶Ⅳ、DNA旋转酶共同改变引起的.实验结果证明,在一定条件下,耐药性的高低与突变位点的多少成正比.     

食品动物源沙门菌aac(6')-Ib-cr基因检测

为检测质粒介导的喹诺酮类耐药性aac(6')-Ib-cr基因在食品动物源沙门菌的分布状况.采用PCR结合BstC1酶切法,以及DNA测序法检测316株食品动物源沙门菌中aac(6')-Ib-cr基因;微量肉汤稀释法检测aac(6’)-Ib-cr阳性和阴性菌株对10种抗菌药物的耐药性;采用PFGE分析阳性菌株的亲缘关系.结果是aac(6')-Ib-cr基因检出率15.82％(50/316);aac(6')-Ib-cr阳性株对氨基糖苷类耐药率高于阴性株,但对环丙沙星、恩诺沙星和氧氟沙星的耐药率低于阴性株;aac(6’)-Ib-cr阳性菌株具有不同的PFGE谱型.结论:aac(6’)-Ib-cr基因在本次检测的食品动物源沙门菌中普遍存在,以水平和垂直传播方式在菌群间传播.     

利用SCOTS技术筛选鸡白痢沙门菌感染巨噬细胞的转录序列

沙门菌在巨噬细胞内的存活是其在宿主内长期定植的主要因素之一,研究利用选择性捕获转录技术(SCOTS)筛选鸡白痢沙门菌S06004株在感染鸡巨噬细胞系HD-11过程中细菌表达的基因.结果显示:鸡白痢沙门菌以MOI值为100:1感染HD-11细胞1h后,感染率达11.75％;选取感染1h时间点作为研究对象,利用SCOTS技术获得了12个鸡白痢沙门菌的转录序列,包括与沙门菌感染相关的毒力基因调控因子invF;构建了invF、tehB和tolC基因插入突变株.突变株侵入HD-11的能力明显低于野生株,反映了SCOTS筛选获得的基因在鸡白痢沙门菌感染巨噬细胞时发挥重要的作用,为深入揭示鸡白痢沙门菌的感染机制提供了基础.     

大肠杆菌多重耐药调节基区AcrR和MarR突变

研究大肠杆菌多重耐药外输泵抑制基因AcrR和MarR突变对大肠杆菌多重耐药的调节机制。采用琼脂平板二倍稀释法测定环丙沙星、诺氟沙星、氯霉素、四环素、利福平、庆大霉素、大观霉素、阿米卡星、链霉素、阿莫西林等10种药物对临床分离的33株大肠杆菌和大肠埃希氏菌的标准菌株ATCC25922的最小抑菌浓度（MIC），从中筛选出7株多重耐药菌和2株相对敏感菌，并对这9株菌及标准菌ATCC25922的AcrR和MarR基因进行聚合酶链式反应（PCR）扩增并克隆后测序，分析DNA序列及氨基酸序列的突变情况。耐药菌和敏感菌均发现有部分菌株发生了不同程度的点突变。AcrR和MarR同时突变将更大限度的提高细菌的耐药性。     

大肠杆菌多重耐药调节基因AcrR和MarR突变

研究大肠杆菌多重耐药外输泵抑制基因AcrR和MarR突变对大肠杆菌多重耐药的调节机制。采用琼脂平板二倍稀释法测定环丙沙星、诺氟沙星、氯霉素、四环素、利福平、庆大霉素、大观霉素、阿米卡星、链霉素、阿莫西林等10种药物对临床分离的33株大肠杆菌和大肠埃希氏菌的标准菌株ATCC25922的最小抑菌浓度(MIC),从中筛选出7株多重耐药菌和2株相对敏感菌,并对这9株菌及标准菌ATCC25922的AcrR和MarR基因进行聚合酶链式反应(PCR)扩增并克隆后测序,分析DNA序列及氨基酸序列的突变情况。耐药菌和敏感菌均发现有部分菌株发生了不同程度的点突变。AcrR和MarR同时突变将更大限度的提高细菌的耐药性。     

Prevalence of the Salmonella plasmid virulence gene "spvD" in Salmonella strains from animals]

Strains of Salmonella isolated from animals in Germany (n = 878) were analysed for the presence of the spvD gene ("Salmonella plasmid virulence gene D") by DNA-DNA hybridization. The spvD gene was only detected in strains of serovars Typhimurium (93.3%), Enteritidis (97.1%), and Dublin (100%) as well as in two rough strains of Salmonella enterica. Salmonella isolates from mammals carried the gene more frequently (cattle 94.0%, horses 92.6%, pigs 73.7%) than those from birds (33.3%) or reptiles (4.5%). Due to its high prevalence in epidemiologically relevant salmonellae, the virulence factor spvD may represent a sensitive and specific target in various serovars for diagnostic or immunization strategies.     

The distribution of Salmonella serovars in chicken and humans in Lithuania

We studied serovars of Salmonella strains isolated from chicken and humans in Lithuania over the period from 2000 to 2004. Salmonella strains were isolated and identified according to the techniques recommended by International Organisation for Standardization (Microbiology of Food and Animal Feeding Stuff--Horizontal Method for the Detection of Salmonella, 1998, ISO, Geneva). The per cent of infected flocks with Salmonella in separate years was between 1.01% and 3.2% during the period of investigation. The contamination rate of broiler legs and breasts was higher (2.36% and 4.25%) than that of wings (0.82%). Eighteen serovars of Salmonella were identified from the total 300 isolated samples. The most prevalent serovars in chicken were Salmonella Enteritidis, Salmonella Infantis and Salmonella Typhimurium. Other serovars such as Salmonella Montevideo, Salmonella Djugu, Salmonella Isangi, Salmonella Bovismorbificans, Salmonella Mbankada, Salmonella Hadar were detected only in one to two samples. In general, similar serovars of Salmonella were found in humans and chicken (S. Enteritidis and S. Typhimurium), although distinct serovars were found only in humans or only in chicken. Analysis of the distribution of Salmonella serovars in humans during the seasons of the year indicated that the highest incidence of Salmonella was in Summer and in the beginning of Autumn. Analysis of the distribution of serovars during the study period indicated that there is a shift over time in both humans and chicken.     

Drug resistance and biochemical characteristics of Salmonella from turkeys.

A study was conducted to determine the antibiotic resistance and biochemical characteristics of 2690 Salmonella strains belonging to 52 serovars and isolated from environmental and feed samples from 270 turkey flocks in Canada. Resistance of the Salmonella strains to the aminoglycoside antibiotics varied widely; none of the strains were resistant to amikacin, 14.2% were resistant to neomycin, 25.8% were resistant to gentamicin, and 27.7% of the strains were resistant to kanamycin. Most strains (97.6%) were resistant to the aminocyclitol, spectinomycin. Regarding resistance to the beta-lactam antibiotics, 14.3% and 14.4% of the strains were resistant to ampicillin and carbenicillin, respectively, whereas only 5 (0.2%) of the strains were resistant to cephalothin. None of the strains were resistant to the fluoroquinolone ciprofloxacin or to polymyxin B. Resistance to chloramphenicol and nitrofurantoin was found in 2.4% and 7% of the strains, respectively. Only 1.7% of the strains were resistant to the trimethoprimsulfamethoxazole combination, whereas 58.1% were resistant to sulfisoxazole. Thirty-eight percent of the strains were resistant to tetracycline. Salmonella serovars differed markedly in their drug resistance profiles. Biochemical characterization of the Salmonella showed that the S. anatum, S. saintpaul and S. reading serovars could be divided into distinct biotypes.     

Antimicrobial resistance pattern of Salmonella serotypes isolated from apparently healthy slaughtered camels (Camelus dromedarius) in eastern Ethiopia

A total of 714 samples consisting of faeces, mesenteric lymph nodes, liver, spleen, abdominal and diaphragmatic muscles (each 119) were collected from November 2001 to April 2002 from apparently healthy slaughtered camels (Camelus dromedarius) in eastern Ethiopia. One hundred sixteen (16.2%) Salmonella strains belonging to 16 different serovars were isolated. All Salmonella strains isolated were examined for antimicrobial resistance to 17 selected antimicrobials. The minimum inhibitory concentration (MIC) values were determined by the microdilution broth test. Fifty-two (44.8%) of the Salmonella isolates were resistant to one or more antimicrobials. Thirty-nine of the 52 (75%) resistant Salmonella serovars exhibited multiple resistance to up to eight different antimicrobials. Among the serovars tested, S. Typhimurium, S. Heidelberg, S. Braenderup and S. Hadar displayed multiple resistance mainly to streptomycin (35.3%), spectinomycin (28.4%), sulfamethoxazole (25.0%), ampicillin (24.1%), trimethoprim (22.4%), trimethoprim/sulfamethoxazole (18.9%), tetracycline (12.9%) and colistin (11.2%). All Salmonella strains tested were susceptible to ciprofloxacin, nalidixic acid, gentamicin, kanamycin and neomycin. The present study showed the importance of camels as a potential source of single and multiple resistant Salmonella strains to different antimicrobials that are also used in the public health sector for the treatment of different bacterial diseases in Ethiopia.     

Loop-mediated isothermal amplification for the rapid, sensitive, and specific detection of the O9 group of Salmonella in chickens

In the present study, the loop-mediated isothermal amplification (LAMP) assay was developed to amplify the fragments of the O9 Salmonella-specific insertion element and evaluated in the laboratory for its potential use in a field situation, such as poultry farms. Among the bacteria tested, a positive reaction was observed only for 128 strains of 6 serovars of the O9 group Salmonella, such as Enteritidis (SE) and Pullorum. The detection limit of the LAMP assay was 10(3)CFU/ml, which was more sensitive than that of the polymerase chain reaction (PCR) assay with the same target gene (10(6)CFU/ml). The final results were obtained within 30 min for the LAMP assay, while the PCR assay needed a total of 120 min. When the LAMP assay was applied to the enrichment broth mixed with cecal dropping samples either spiked with SE in vitro or excreted by SE-inoculated hens, the results were comparable to those of the conventional plating method including 2 separate enrichments. In conclusion, the LAMP assay developed in the present study is an effective method for the specific detection of the O9 group Salmonella serovars, including SE.