污水厂产超广谱β内酰胺酶大肠杆菌通过接合水平传递耐药性

【目的】研究废水中产超广谱β-内酰胺酶大肠杆菌中可移动质粒在耐药基因水平传播机制中的作用。【方法】对污水厂分离所得的50株产ESBLs大肠杆菌进行接合试验,并对所得的接合子采用纸片扩散法测定其对15种常见药物的耐药表型,针对质粒介导的产ESBLs菌株的耐药基因设计7对特异性引物对接合子进行PCR扩增。【结果】研究结果显示,80份水样分离得50株产ESBLs大肠杆菌,共接合成功35株细菌,接合成功率高达70%。接合子与供体菌相比,均发生耐药谱型的改变,且存在丢失一种或几种药物耐药性且产生另一种或几种药物耐药性的现象。PCR扩增结果显示,接合子与供体菌相比,耐药基因型有所减少或不变,bla_(TEM)、bla_(CTX-M)基因全部接合成功,bla_(SHV)基因仅1株未接合成功,耐氟喹诺酮类基因未发生转移。【结论】本研究表明,不同的耐药基因可能位于不同的可移动质粒上,可移动质粒在大肠杆菌耐药性水平传播的过程中起到了十分重要的作用。     

5种大肠埃希氏菌Col质粒接合传递实验研究

以5种61株Col~ 大肠埃希氏菌为供体,与受体E.Coli K_(12)进行接合实验,结果有16株供体菌的Col质粒传递给K_(12),占25.4％。其中NPEC、ETEC、EPEC、EIEC、EAEC的传递比率分别是8/22、 4/21、2/15、1/2、1/1。按传递方式分类,52株耐药供体菌有10株的Col质粒和耐药标记(或R质粒)发生共同传递,占19.2％,伴发Col质粒传递的耐药标记有8种,其中TC、SM、COS、KM的并发传递率较高(16—20％)。不同耐药标记菌株的Col质粒传递率范围在0—66.7％。在8株敏感菌和1株耐药菌中有6株Col质粒发生单独传递,占66.7％。实验发现4个配对组的Col质粒和R(r)质粒发生分离而独自传递。本文分析了Col质粒传递方式和耐药性的关系,同时讨论了接合传递的不同方法。为阐明和研究R~ Col~ 肠道菌的演变及其对正常菌群生态平衡的影响提供遗传学依据。     

大肠埃希菌耐药性监测及耐药质粒的研究

为了解医院内感染大肠埃希菌的耐药情况,探讨其耐药发生、流行及传播机制,对从临床分离的32株大肠埃希菌进行了药敏试验、质粒图谱分析以及质粒接合、转化试验,结果表明大肠埃希菌对氨苄青霉素的耐药率＞88%,对庆大霉素的耐药率＞75%,其中28株大肠埃希菌检出质粒,均含有一条分子量为5.66 Mu的质粒带,是医院内感染大肠埃希菌的流行质粒.质粒的接合、转化试验证实了质粒具有横向传播的特点,是细菌产生耐药的主要原因.     

多重耐药阴沟肠杆菌质粒型AmpC酶DHA-1基因的检出

目的测定56株同时耐头孢噻肟、庆大霉素和环丙沙星的阴沟肠杆菌质粒型AmpC酶基因型。方法先后用头孢西丁纸片法、三维试验、等电聚焦及酶抑制试验和微量稀释法进行表型检测。接合试验证实酶基因的转移性。多重聚合酶链反应以及基因测序等方法鉴定质粒型AmpC酶基因型。结果受试的56株细菌中有5株三维试验阳性,其中1株能转移接合,接合子多重聚合酶链反应扩增结果呈阳性,等电点为7．8,基因测序表明和DHA-1型AmpC酶一致。结论我国的多重耐药阴沟肠杆菌已获得质粒型DHA基因,DHA基因可通过转化、接合等方式转移给其他细菌且易于传播,因此应加强监控以防DHA基因在革兰阴性菌中流行。     

食源性沙门氏菌耐药性检测及相关质粒

[目的]测定390株沙门氏菌的抗生素药敏性,研究部分多重耐药株中质粒与其宿主耐药表型之间的关系及其在接合过程中对耐药性水平转移的影响.[方法]使用选择性培养基分离到沙门氏菌并通过PCR确认后,按照琼脂稀释法测定分离株对供试抗生素的药敏性,试剂盒提取代表性多重耐药株中的质粒,HindⅢ酶切,DPS软件分析电泳后质粒图谱.通过接合试验研究质粒在抗生素抗性水平转移中的作用.[结果]沙门氏菌分离株对四环素耐药最为普遍(58.2%),其次为链霉素(42.8%)、卡那霉素(39%)和氨苄青霉素(38.2%),对头孢西丁、氯霉素、庆大霉素、头孢曲松、阿莫西林甲氧苄啶、头孢替呋钠和萘啶酮酸的耐药率分别为27.2%、26.9%、21%、19%、18.2%、17.9%、14.6%和12.3%.抗性质粒编码的相同或相似沙门氏菌耐药表型与其中所含的耐药质粒并不呈现出严格的对应关系.质粒携带的抗性基因可通过接合作用转移,接合效率在2.4×10-4到5.6×10-1之间.[结论]食源性沙门氏菌对常用抗生素的多重耐药已经成为普遍现象,抗性质粒的同源性与其宿主耐药表型无直接相关性,其携带的耐药基因可通过接合作用在不同细菌种属之间高频传递.     

江苏部分地区食源性和人源沙门氏菌的多重耐药性研究

从江苏省部分地区收集了117个沙门氏菌分离株,其中食物源和人源菌株分别有81株和36株。16种抗生素敏感性试验表明,有111个分离株对2种或2种以上的抗生素有耐药性,人源沙门氏菌分离株的抗生素耐药率比食物源的高,单一抗生素以链霉素耐药率(92.3%,108/117)最高。对5种或5种以上抗生素耐药的分离株有59株(50.4%),其中对特定六种抗生素:氨苄青霉素、氯霉素、链霉素、磺胺、四环素和卡那霉素耐药(ACSSuTK,R型)的菌株有12株。设计18对耐药基因和I类整合子保守区的引物,对36株有不同来源和耐药特征的多重耐药菌株进行耐药基因和I类整合子的检测,PCR扩增结果与抗生素敏感性表型一致。有30株细菌携带有I类整合子,大小为0.3、0.6、1.0、1.2和1.6kb,其中1.6kb(aadA5-dfr17)大小的整合子在25株细菌中分布(24/36)。接合试验表明,氨苄青霉素、氯霉素、链霉素、甲氧苄氨嘧啶和四环素的耐药特性是由接合性质粒携带。结果显示,耐药基因多数由I类整合子和质粒携带,可以通过接合试验发生转移,可移动的DNA成分可能在耐药特性的转移和分布中起到重要作用。     

产AmpC酶肺炎克雷伯菌对氨基糖苷类抗生素的耐药性分析

目的了解本地区质粒介导的耐药机制在产AmpC酶肺炎克雷伯菌多重耐药中的作用、氨基糖苷修饰酶(AMEs)基因类型及转移方式。方法头孢西丁三维试验方法,检测产AmpC酶菌株;采用接合转移试验了解肺炎克雷伯菌耐药基因转移方式。采用K-B纸片测定产AmpC接合子对4种氨基糖苷类抗生素的敏感性,并采用PCR技术检测AMEs基因。结果临床分离的820株肺炎克雷伯菌共筛选出108株疑产AmpC酶菌株,阳性率为13.17%。经接合转移试验、AmpC酶表型确认试验共获得53株产AmpC接合子。其中67.9%的接合子(36/53)检出氨基糖苷修饰酶基因,aac(3)-I和aac(6′)-II未检出,对4种常用氨基糖苷类抗生素(阿米卡星、庆大霉素、妥布霉素、奈替米星)耐药率分别为37.7%、68.0%、43.4%和62.3%。结论本地区质粒介导AmpC酶是肺炎克雷伯菌产生多重耐药的重要原因,产酶株对氨基糖苷类高度耐药,其耐药性与AMEs密切相关,耐药基因质粒的转移可导致耐药性的传播扩散。     

Tn2促进基因缺失与R质粒接合转移有关的遗传学证据

通过R100-1的接合转移和非接合转移的温度选择两种方法分别获得了一系列mer::Tn2突变体。回复试验和杂交结果表明,通过接合转移分得的22株mer::Tn2突变体中有7株发生了长度不等的缺失;但通过非接合转移的温度选择法获得的17株mer::Tn2突变体中没有发现缺失株。这一结果进一步证明Tn2促进缺失形成与R因子的接合转移有关。     

亚抑菌浓度头孢西丁对耐药质粒接合转移的影响

目的探讨亚抑菌浓度头孢西丁对耐药质粒接合转移的影响,研究病原菌耐药性播散的产生机制。方法采用聚合酶链反应（PCR）分析临床分离的63株产ESBLs肺炎克雷伯菌株SHV型β-内酰胺酶编码基因。质粒接合转移试验采用肉汤接合法。研究不同亚抑菌浓度头孢西丁（0、1、0.5、0.25、0.125μg/ml）和不同作用时间（2、4、6、8、10、12 h）下临床产ESBLs肺炎克雷伯菌株供体菌与受体菌E.coliC600接合转移频率的变化。结果63株临床分离的产ESBLs肺炎克雷伯菌株有41株扩增出SHV型基因,阳性率为65.08%。随着亚抑菌浓度头孢西丁作用时间的增加,接合转移频率有随之增加的趋势。在相同作用时间下,头孢西丁浓度0.125μg/ml作用下的接合转移频率高于其他亚抑菌浓度的作用。结论应合理使用抗菌药物,减少抗菌药物的选择性压力,防止耐药细菌传播。     

Tn5-Mob系统诱导根瘤菌属之间耐盐和共生性状的转移

本实验通过质粒pSUP501l及其辅助质粒RP4将Tn5-Mob随机插入苜蓿根瘤菌(Rhizo-bium meliloti 042B)的基因组中,得到86株接合子SR。随机选取4株SR,通过辅助质粒R68．45的三亲本杂交,将它们的DNA片段引入慢生型大豆根瘤菌(Bradyrhizobium,japo—nicum USDA 110),获得106株接合子BSR。大部分BSR菌株获得了生长快速的特性和耐盐性,一般能耐0．3—0．5m01／L Nacl,其中有些菌株能产生黑色素。将9o株BSR回接大豆和苜蓿植株,发现47株能在大豆和苜蓿植株结瘤,但在苜蓿卜无固氮活性;26株只能在大豆植株结瘤固氮;13株只能在苜蓿植株结瘤而不固氮;4株在大豆和菖蓿植株均不结瘤。其中,获得了4株耐盐性和固氮酶活性强的接合子。     

Drug resistance of the causative agent of Grigo'rev-Shiga dysentery (Shigella dysenteriae 1)isolated in the USSR

Forty strains of S. dysenteriae 1 isolated in the USSR within 1986-1988 were tested for their resistance to 11 antibacterial drugs. It was shown that 92.5-97.5 per cent of the isolates were resistant to chloramphenicol (Cm) and tetracycline (Tc), 22.5 per cent to streptomycin (Sm), 17.5 per cent to nalidixic acid (Nal) and 10 per cent to ampicillin (Ap) and polymyxin (Pm). Resistance to Cm Tc (51.4 per cent) and Cm Tc Nal (13.5 per cent) represented the predominating phenotype. 35 per cent of the strains carried conjugative R plasmids. In the majority of the cases, the determinants of resistance to Cm and Tc were transferred, which must define the high frequency of the strains resistant to Cm and Tc. All the tested strains were sensitive to gentamicin, neomycin, rifampicin, cefamezin and ciprofloxacin. Since the strains of S. dysenteriae 1 proved to be highly sensitive to the tested drugs it appeared possible to consider them as the drugs of choice in etiotropic therapy of patients with dysentery caused by the pathogens of the Grigoryev-Shiga group.     

Antibiotic sensitivity and the characteristics of transmissive R plasmids in Shigella belonging to various species]

Sensitivity of 241 Shigella strains isolated from patients at various regions of the USSR in 1975--1978 was tested with respect to 14 antibiotics by the method of serial dilutions. 90.5 per cent of the isolates proved to be resistant to the antibacterial drugs and the greater part of 75.9 per cent of them had multiple resistance. The resistance of the Shigella was most pronounced and frequent with respect to tetracycline, streptomycin, levomycetin, as well as ampicillin and carbenicillin. Gentamicin, cephaloridin, polymyxin M, kanamycin, monomycin, neomycin and rifampicin were highly active against the Shigella. More than 50 per cent of the isolates were sensitive to levomycetin, ampicillin and carbenicillin. Differences in the frequency of the resistant strains and the spectrum of the antibiotic resistance of different Shigella subgroups (species) were observed. The study of 173 multiple resistant Shigella strains showed that about 67 per cent of the strains had a capacity for transduction of the resistance markers into the recipient cells of E. coli. The conjugative R-plasmids were most frequent in Sh. boydii and Sh. sonnei (95 and 95 per cent respectively), less frequent in Sh. flexneri and Sh. newcastle (68 and 53 per cent respectively) and least frequent in the mannitol negative Shigella (25 per cent). The capacity for transduction of R-plasmids in the strains carrying the determinants of resistance to 2 or 3 antibiotics was higher than in the strains carrying the determinant of resistance to one antibiotic. The clinical Shigella strains tested mainly had transmissive R-plasmids of fi--character (79 per cent).     

R-plasmids of bacteria of the genus Pseudomonas isolated from industrial sewage

A total of 132 Pseudomonas strains isolated from untreated sewage of antibiotic plants were tested. A significant number of the strains were resistant to streptomycin (77 per cent), carbenicillin (75 per cent), kanamycin (37.5 per cent) and tetracycline (23 per cent). Eighteen conjugative and 3 nonconjugative resistance plasmids were detected in 19 strains. The genes determining the resistance to streptomycin, kanamycin and tetracycline were most frequent. The frequency of the plasmid transfer between the strains of Ps. aeruginosa (PAO) varied within 10(-3)--10(-7) per donor cell. Six plasmids belonged to group Inc P-1. Four plasmids belonged to group Inc P-2, 3 plasmids to groups Inc P-3 and Inc P-5 and 1 plasmid to group Inc P-7.     

Possible dependence of the results of phage typing Ps. aeruginosa strains on the presence of R plasmids

The plasmid composition of 209 strains of Ps. aeruginosa was determined. The strains were isolated from patients, animals and environment in different geographical areas. The number of the plasmid-containing strains averaged 26.8 per cent. The molecular weights of the plasmids varied from less than 10 to more than 150 MD. 41 conjugative plasmids were transmitted to the recipients of Ps. aeruginosa RAO 303. 66 per cent of them had a restrictive effect on the development of phages used in genetic studies, epidemiological phage typing (Lindberg Collection), and medical practice. This resulted in the changing of the phage type of the host strain. Similar results were obtained in the studies with 10 standard R plasmids representing different incompatibility groups. No relation between the spectrum of the phage restriction, group specificity and the other properties of the plasmids was observed. About 50 per cent of the plasmids markedly lowered the sensitivity level of Ps. aeruginosa RAO 303 to the therapeutic pyocyanic phage. The systems of restriction and modification of DNA coded with plasmids were not detected. A possible changing of the phage type of Ps. aeruginosa strains under the effect of R plasmids should be considered in epidemiological assays and respective treatment measures.     

Antibiotic resistance and its transmissibility in strains of opportunistic enterobacteria

Sensitivity of 39 opportunistic Enterobacteria strains was studied with respect to tetracycline, kanamycin, ampicillin, levomycetin, streptomycin and nevigramon. The strains were isolated from children with acute intestinal infections of obscure etiology. 38 isolates were resistant to one or more drugs. The highest number of the strains was resistant to kanamycin, ampicillin and levomycetin, i.e. 81.5, 84.2 and 84.2 per cent of the cultures respectively. Nevigramon proved to be the most effective in vitro (76.4 per cent of the sensitive strains). The ability of the strains to transfer the antibiotic resistance markers on conjugation was tested. Transmission of R plasmids was shown in 75 per cent of the isolates.     

Rate of plasmid transfer among Escherichia coli strains isolated from natural populations.

The conjugative plasmid R1 was introduced into ten strains of Escherichia coli isolated from natural populations. Spontaneous nalidixic-acid-resistant mutants of the ten strains served as recipients. The ten donor and recipient strains were mated in all combinations and the rate at which R1 transferred between the strains was determined. The rate of transfer ranged from 5.2 x 10(-11)-1.1 x 10(-18) ml per cell h-1, and averaged 1.3 x 10(-15) ml per cell h-1. The results of these experiments suggest that the rates of conjugative transfer are far too low for plasmids to be maintained as parasites in their host populations. Infectious transfer is insufficient; plasmids must confer a selective advantage to their host to be maintained.     

R plasmid distribution in S. typhi isolated on the territory of the left coast of the Ukraine in 1970-1982

A total of 373 strains of S. typhi isolated in 1970-1982 were tested with respect to their sensitivity to 9 antibiotics active against gram-negative bacteria. It was shown that about 1/3 of the isolates were resistant to 1-3 antibiotics. Among the resistant isolates the number of strains resistant to 4-5 and more antibiotics amounted to 12.5 per cent. The plasmid nature of the antibiotic resistance in the isolated strains of S. typhi was shown. Transmissive R plasmids were detected in 13 per cent of the strains studied. Within the last 5 years there was an increase in the proportion of strains with transmissive R plasmids in patients with sporadic typhoid fever or especially in groups of patients with the disease.     

R plasmids among Gram-negative bacteria with multiple drug resistance isolated in a general hospital.

The incidence of conjugative R plasmids in multiple drug-resistant strains of gram-negative bacteria isolated in 1973 from patients in a 700-bed general hospital in Tokyo and some properties of the R plasmids isolated are described. Conjugative R plasmids were found in 52 of the 96 strains (54%), from which 74 R plasmids were demonstrated. It is remarkable that the isolation frequency of R plasmids mediating quadruple- or five-drug resistance was rather low, and the complete pattern of multiple resistance of the original isolates was only rarely transferred by conjugation. These results revealed the existing state of the distribution of R plasmids among hospital strains with multiple drug-resistance.     

Activities and survival of endophytic bacteria in white clover (Trifolium repens L.)

In this study, the genera, abundance, and activities of endophytic bacteria in field-grown white clover (Trifolium repens) and the fate of introduced antibiotic-tolerant bacteria in white clover tissues were investigated. Pseudomonas, Pantoea, and Corynebacterium were the most frequently isolated endophytic bacteria genera, whereas Xanthomonas, Microbacterium, and Cellulomonas occurred less frequently. The average bacterial populations in stolons and roots were approximately 100,000 colony-forming units (CFU) (g wet mass)-1. Of the 28 strains tested for activity, none were chitinolytic or able to inhibit the root pathogen Codinaea fertilis in vitro. However, Fusarium oxysporum and Cylindrocladium scoparium were inhibited by one and five strains, respectively. Four of seven strains tested depressed clover seedling growth. In pot experiments, colonization and recovery of spontaneous rifampicin-tolerant mutants (Rif+) of bacteria were studied in clover plants for periods up to 20 weeks. The strains used, sourced from white clover (endophytic and rhizoplane) and organic compost, had previously shown growth promotion potential of white clover seedlings by increasing plant mass and decreasing nematode numbers. In one experiment in this present study, five Rif+ strains were individually inoculated onto white clover seedlings, all five were re-isolated from shoots after 6 weeks and four strains were re-isolated after 20 weeks (numbers of Rif+ bacteria ranged from 51 to 200 CFU (g wet mass)-1). No Rif+ bacteria were isolated from root tissue at either time. In the second experiment, conducted with two strains of Rif+ bacteria, the population was highest in the shoots (range>500 CFU of Rif+ bacteria (g shoot fresh mass)-1) in weeks 2 and 3, declining to <200 CFU in week 5. Again, no Rif+ bacteria could be detected in roots. No Rif+ bacteria were recovered after 14 weeks for one of the strains. It appears that the main route of bacterial entry into seedlings was through stomata and that bacteria remained in the aerial parts of plants rather than migrating to the roots.