Characteristics of conjugative R-plasmids from pathogenic avian Escherichia coli.

Three of four virulent avian Escherichia coli isolates transferred a single large molecular-weight R-plasmid to two recipient E. coli strains. Antibiotic resistances transferred included streptomycin (two isolates) and streptomycin-tetracycline-sulfa (one isolate). Production of colicin and siderophores, complement resistance, and embryo lethality present in the virulent isolates were not transferred to recipient organisms. From the results, it appears that the R-plasmids of these virulent avian E. coli are not associated with virulence.     

The Ins and Outs of siderophore mediated iron uptake by extra-intestinal pathogenic Escherichia coli

Extraintestinal pathogenic E. coli (ExPEC) are responsible for many infectious diseases in livestock, such as airsacculitis in poultry, acute mastitis in dairy animals and neonatal septicaemia and urinary tract infections (UTI) in pigs and cattle. In their animal hosts, ExPEC have to cope with low iron availability. By using different strategies, ExPEC strains are able to retrieve iron sequestered by host proteins. One of these strategies is the use of siderophores, which are small secreted molecules with high affinity for iron. ExPEC are known to synthesize up to four different types of siderophores: enterobactin, salmochelins, yersiniabactin and aerobactin. Steps required for iron acquisition by siderophores include (1) siderophore synthesis in the cytoplasm, (2) siderophore secretion, (3) ferri-siderophore reception, (4) ferri-siderophore internalization and (5) iron release in the cytoplasm. Each siderophore has specific properties and may be differentially regulated to provide different advantages, potentially allowing ExPEC to adapt to different environmental conditions or to overcome host innate immunity. Iron acquisition by siderophores plays a significant role in ExPEC virulence and, as it requires outer membrane receptors, it constitutes an interesting target for the development of vaccines that could be used to limit the number of infectious diseases due to ExPEC in livestock.     

The expression of plasmid mediated afimbrial adhesin genes in an avian septicemic Escherichia coli strain

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD₅₀ of 4.0 × 10⁵ CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD₅₀ of 1.2 × 10¹² CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes.     

Location of increased serum survival gene and selected virulence traits on a conjugative R plasmid in an avian Escherichia coli isolate

Avian colibacillosis is a costly disease for the poultry industry. The mechanisms of virulence employed by the etiologic agent of this disease remain ill defined. However, accumulated evidence suggests that complement resistance and the presence of the increased serum survival gene (iss) in an avian Escherichia coli isolate may be indicative of its ability to cause disease. This association of iss with the E. coli implicated in avian disease may mean that iss and/or, perhaps, the genes associated with it are important contributors to avian E. coli virulence. For this reason, we have begun a search for iss's location in the bacterial genome. Thus far, iss in an avian E coli isolate has been localized to a conjugative R plasmid and estimated to be about 100 kilobase (kb) in size, encoding resistance to tetracycline and ampicillin. Hybridization studies have revealed that this plasmid contains sequences with homology to tsh, a gene associated with virulence of avian E coli; intI 1, a gene encoding the integrase of Class 1 integrons; and certain genes of the aerobactin- and CoIV-encoding operons. Sequences homologous to merA, a gene of the mercury resistance operon, were not identified on this R plasmid. This plasmid, when transferred into an avirulent, recipient strain by conjugation, enhanced the transconjugant's resistance to complement but not its virulence, in spite of the plasmid's possession of several putative virulence genes and traits. Such results may reflect the multifactorial nature of virulence, the degree of the recipient's impairment for virulence, or an inability of the embryo assay used here to detect this plasmid's contribution to virulence. Additionally, this plasmid contains genes encoding antimicrobial resistances, which may provide a selective advantage to virulent E. coli in the production environment. Further study will be needed to determine whether this plasmid is widespread among virulent E. coli and to ascertain the implications that this link between virulence and antimicrobial resistance genes may have for poultry management.     

大肠埃希氏菌多重耐药性与整合子的研究

整合子目前被认为是耐药基因在水平传播的重要因子,由Hall等命名,由两部分组成,5'与3'端保守区域(conserved segments简称cs)以及中间的基因盒,具有特异重组位点,能够将携带有耐药基因的基因盒选择性地整合到整合子上,获得耐药性[1].     

Multiple antimicrobial resistance region of a putative virulence plasmid from an Escherichia coli isolate incriminated in avian colibacillosis

Infections due to Escherichia coli have been costly to the poultry industry, but the exact virulence mechanisms used by these organisms to cause disease in birds remain undefined. Several factors have been shown to contribute to the virulence of avian E. coli, and many of the genes encoding these factors have been found on large conjugative plasmids. Because of the occurrence of antimicrobial resistance genes on these same plasmids, it is possible that the use of antimicrobial agents may select for persistence of E. coli containing such plasmids. In the present study, a subclone of one of these plasmids was identified as likely containing some virulence and antimicrobial resistance genes. In an effort to better understand the relationship between virulence and resistance in these plasmids, this subclone was sequenced and the sequence analyzed. Analysis of this 30-kilobase (kb) region of plasmid pTJ100 revealed a mosaic of virulence genes, insertion sequences, antimicrobial resistance cassettes, and their remnants. Many of the resistance genes found in this region were expressed under laboratory conditions, indicating that certain antimicrobial agents, including disinfectants, antibiotics, and heavy metals, could promote selection of E. coli containing such plasmids in the production environment. Also, analysis of the G + C content of this clone indicated that it is the likely consequence of a complex evolution with components derived from various sources. The occurrence of many mobile elements in conjunction with antimicrobial resistance and virulence genes in this 30-kb region may indicate that the genetic constitution of the clone is quite plastic. Although further study will be required to better define this plasmid's role in avian E. coli virulence, the sequence described here is, to our knowledge, the longest known contiguous sequence of a ColV plasmid yet presented. Analysis of this sequence indicates that this clone and its parent plasmid may be important to the pathogenesis of avian colibacillosis and the evolution of avian E. coli virulence.     

Relationship of complement resistance and selected virulence factors in pathogenic avian Escherichia coli.

Complement resistance, antibiotic resistance profiles, and virulence profiles of 80 Escherichia coli isolates from the intestines of normal chickens (40 isolates) and chickens diagnosed as having colisepticemia (40 isolates) were compared. Differences were observed between the two groups for antibiotic resistance, siderophore production, presence of type 1 pili, complement resistance, motility, and size of plasmids. The systemic isolates were more likely to have siderophores and type 1 pili, and to be complement-resistant and motile than were the intestinal isolates. No differences between the two groups were observed for colicin production. Further comparison of the 10 most complement-resistant isolates from the systemic group and 10 most complement-sensitive isolates from the intestinal group revealed a correlation between an isolate's resistance to complement and its ability to kill embryos, express type 1 pili, and be motile. Virulence of avian E. coli strains appears to be correlated with complement resistance and the interaction of this resistance with the ability to produce type 1 pili and be motile.     

53株禽致病性大肠杆菌的耐药表型及耐药基因的检测

为了解禽致病性大肠杆菌耐药表型及耐药基因的情况,选取江苏、安徽等地分离的53株禽致病性大肠杆菌,采用药敏纸片法对9种抗菌药物进行药敏试验,并对四环素类tet(A)、tet(B)、tet(C)、tet(W)、tet(M)、tet(O)、tet(K)、tet(L)耐药基因,喹诺酮类GryA、ParC耐药基因,磺胺类sulⅠ、sulⅡ、sulⅢ耐药基因,β-内酰胺类SHV、CTX-M、ompCA耐药基因,氨基糖苷类aac(3)-Ⅰ、aac(3)-Ⅱ、aac(6')-Ⅰb、aac(6')-Ⅱ、ant(3″)-Ⅰ、armA、rmtA、rmtB、rmtC、rmtD、npmA耐药基因进行PCR检测。结果显示:53株禽致病性大肠杆菌对磺胺异噁唑、环丙沙星、氨苄西林、多西环素的耐药率较高,分别为88.68%(47/53)、71.71%(38/53)、86.79%(46/53)、75.47%(40/53)。其中50株禽致病性大肠杆菌表现为多重耐药,耐4、5、6种药物的现象最为普遍,且不同地区菌株存在差异。tet(A)是四环素耐药基因中最为流行的一种耐药基因(52.83%,28/53),喹诺酮类耐药基因主要由gryA(94.33%,50/53)、parC(94.33%,50/53)基因编码,耐磺胺类药物sulⅠ、sulⅡ、sulⅢ基因均有检出,分别为96.23%(52/53)、98.11%(48/53)、86.79%(46/53),耐β-内酰胺类药物中仅检出ompC A基因(30.19%,16/53),在检测的11种耐氨基糖苷类耐药基因中,最为流行为aac(3)-Ⅱ、aac(6')-Ⅰb、ant(3″)-Ⅰ基因,分别为92.59%(49/53)、98.11%(52/53)、100%(53/53)。耐药基因与相关耐药菌株检出率基本呈正相关。试验结果表明:53株禽致病性大肠杆菌耐药性高,耐药谱广,耐药基因流行现象十分普遍。本试验结果能为禽致病性大肠杆菌的耐药现状与临床用药提供理论指导。     

Serum resistance and virulence of Escherichia coli isolated from turkeys

Twenty-five strains of Escherichia coli isolated from turkeys were characterized for their serum resistance and virulence. An in vitro bactericidal assay was used to determine the serum resistance of E coli. Virulence was determined by survival time after IV inoculation of each strain into 3-week-old turkeys. Serum-resistant E coli strains were generally found to be virulent for turkeys, whereas serum-sensitive E coli strains were avirulent. Of the 25 strains, 18 strains were placed in the 2 categories of serum-resistant/virulent and serum-sensitive/avirulent. Five strains were serum-resistant and avirulent, and 2 strains were serum-sensitive and virulent. Serum resistance appears to be an important determinant of virulence for E coli in turkeys; however, the requirement for other virulence factors, in addition to serum resistance, was suggested by the finding that 5 serum-resistant strains were avirulent in turkeys.     

Serum resistance and the traT gene in bovine mastitis-causing Escherichia coli

Ninety-five Escherichia coli isolates from bovine mastitis, 47 isolates from milking machine filters, 36 enterotoxigenic (ETEC) and 43 verocytotoxigenic (VTEC) isolates from cows were examined for the ability to resist the bactericidal effects of 90% gnotobiotic calf serum. There was no significant difference in the percentage of isolates in each group which demonstrated resistance. Two potential virulence traits, the traT gene and the K1 capsular antigen, previously shown to be related to serum resistance, in human E. coli pathogens, were also examined. Using colony blot hybridization there was no significant difference in the percentage of isolates in each group carrying the traT gene. A significant relationship between the presence of the traT gene and serum resistance was not found in any of the four groups of E. coli isolates tested. Only 3.2% of the bovine mastitis, 2.1% of the milk filter and 4.6% of the VTEC isolates were positive for the K1 capsular antigen. Again, no correlation between either the K1 antigen and serum resistance or between the K1 antigen and the presence of the traT gene was found in any of the four groups. None of the antimicrobial resistance patterns of the isolates were the same as those demonstrated by R plasmids known to carry the traT gene. Thus, it appears that the traT gene may not be related to serum resistance in bovine E. coli isolates.     

Quinolone resistance in Escherichia coli.

Escherichia coli is an important pathogen of animals and humans that causes great financial cost in food production by causing disease in food animals. The quinolones are a class of synthetic antimicrobial agents with excellent activity against Escherichia coli and other Gram-negative bacteria used in human and veterinary medicine. Different quinolones are used to treat various conditions in animals in different parts of the world. All members of this class of drug have the same mode of action: inhibition of topoisomerase enzymes, DNA Gyrase and Topoisomerase IV. Escherichia coli can become resistant to quinolones by altering the target enzymes, reducing permeability of the cell to inhibit their entry, or by actively pumping the drug out of the cell. All these resistance mechanisms can play a role in high-level fluoroquinolone resistance, however target site mutations appear to be most important. As all quinolones act in the same way resistance to one member of the class will also confer decreased susceptibility to all members of the family. Quinolone resistant Escherichia coli in animals have increased in numbers after quinolone introduction in a number of different case studies. The resistance mechanisms in these isolates are the same as those in resistant strains found in humans. Care needs to be taken to ensure that quinolones are used sparingly and appropriately as highly resistant strains of Escherichia coli can be selected and may pass into the food chain. As these drugs are of major therapeutic importance in human medicine, this is a public health concern. More information as to the numbers of quinolone resistant Escherichia coli and the relationship between resistance and quinolone use is needed to allow us to make better informed decisions about when and when not to use quinolones in the treatment of animals.     

皖北地区禽源大肠杆菌的血清型鉴定与耐药性分析

为了掌握皖北地区家禽致病性大肠杆菌的优势血清型及耐药性表现,从皖北4个市8个县区32个规模化养殖场采集疑似家禽大肠杆菌病的病料64份,通过常规分离培养纯化获取31株致病性大肠杆菌,对31株致病性大肠杆菌进行血清学鉴定和耐药性分析。试验结果表明:31株致病性大肠杆菌分别属于O_(78)、O_(18)、O_(88)、O_2、O_1、O_(143)、O_4、O_(35)、和O_(141)血清型,其中O_(78)9株、O_(18)7株、O_(88)5株,共计21株,占分离株的67.74%,属于本地区的优势血清型。致病性试验结果表明,所分离的31株致病菌中,83.9%具有高致病性,12.9%具有中致病性,3.2%具有低致病性,不同血清型致病性没有明显差异。分离菌株对11种抗生素表现不同程度的耐药性,对复方磺胺甲基异噁唑、磺胺二甲嘧啶、恩诺沙星表现严重耐药,耐药比例在70%以上;对阿莫西林、链霉素、庆大霉素、卡那霉素、氟苯尼考、环丙沙星耐药比例在16%以上;对头孢噻肟、头孢唑肟、阿米卞星耐药性比例在10%以下。其中,对4、5、6种药物表现耐药比例较高,占分离株的60%以上,对3、7、8、9耐药性居中,所有菌株均表现为多重耐药性。研究结果为皖北地区家禽大肠杆菌病的疫苗防控、药物防治提供了科学依据。     

Adhesive properties and iron uptake ability in Escherichia coli lethal and nonlethal for chicks

Escherichia coli that are virulent for poultry usually result in a respiratory disease, which is frequently followed by a general infection. Adhesiveness of E. coli to epithelial cells and iron-uptake ability of E. coli could be involved in different steps of the disease. These properties were studied in 59 E. coli strains originating from poultry, with reference to lethality for day-old chicks. Adhesive properties were found in 64% of the lethal strains and in only 23% of the nonlethal strains. The ability to grow in limited iron conditions was strongly correlated with lethality. Fifty-two percent of the lethal E. coli strains, but none of the nonlethal strains, possessed both adhesive and iron-uptake abilities. It is suggested that these two properties play a role in the virulence of E. coli for poultry.     

In vivo transfer of an Escherichia coli enterotoxin plasmid possessing genes for drug resistance.

Experiments were conducted to study transfer of an enterotoxin (Ent) plasmid from a porcine enteropathogenic Escherichia coli to an E coli K12 strain in the intestine of newly weaned pigs. The Ent plasmid carried genes for resistance to tetracycline, streptomycin, and sulfonamides, thereby permitting a selection for tetracycline-resistant exconjugants in the feces of the pigs. In vivo transfer of the Ent plasmid was demonstrated to occur when the pigs were given large oral inocula of donor and recipient cultures, 1 hour apart. Differences in extent of transfer were not detected in pigs given antibiotic-free feed compared with littermates on feed containing oxytetracycline at 50 g/ton. In one experiment, tetracycline-resistant Ent- exconjugants were found which appeared to have received an R plasmid from an enteropathogenic type of E coli resident in the intestine.     

禽致病性大肠杆菌ST95流行菌株耐药表型及耐药基因分析

为探究禽致病性大肠杆菌(APEC)ST95类群菌株的耐药表型和耐药基因分布情况,对37株ST95 APEC菌株进行血清型、耐药性测定以及耐药基因检测。结果显示:37株ST95 APEC的优势血清型为O2:K1和O1:K1,所有禽源ST95菌株均为多重耐药(MDR)特性,其至少对12种抗生素耐药,ST95菌株对头孢类抗生素的耐药比率很高,对β-内酰胺酶抑制剂耐药率也达到35%,对非头孢类抗生素如环丙沙星、庆大霉素等同样具有广泛耐药性。PCR检测发现30株APEC菌株含有β-内酰胺酶基因,在这些ST95菌株中检测出多种质粒编码的抗性基因,如链霉素/壮观霉素抗性基因strA和strB等。研究表明ST95 APEC菌株呈现明显的广谱耐药特性,质粒携带的耐药基因广泛分布于ST95菌株。     

Failure of the Congo red dye uptake test to discriminate between virulent and avirulent avian Escherichia coli.

Twenty avian Escherichia coli isolates from normal and diseased chickens were compared by use of three virulence tests. These tests included the uptake of Congo red dye, an embryo lethality test, and a quantitative microtiter complement resistance test. A direct correlation was seen between the results of the complement resistance test and the embryo lethality test. The results of the Congo red test did not correlate with the two other tests.     

Biological characteristics and pathogenicity of avian Escherichia coli strains.

Fifty avian (chicken) pathogenic Escherichia coli strains (APEC) isolated from individuals suffering from omphalitis, septicaemia and swollen head syndrome, and 30 strains isolated from healthy chickens were studied regarding their biological characteristics such as serogroups, haemolysin, colicin, cytotoxin, toxin and siderophore production, adhesion capacity to in vitro cultivated cells, and absorption of Congo red dye. Serotyping demonstrated that most of the omphalitis and normal strains were untypable, whereas most of the septicaemic strains were either untypable or rough. There was no prevalent serogroup among the pathogenic strains studied. The capacity for adhesion and invasion of in vitro cultured cells (HeLa, HEp-2, KPCC), as well as the agglutination of different types of red blood cells and the LD50 of each strain were also evaluated. No correlation was observed between the biological characteristics and pathogenicity, except that colicin was characteristically produced by swollen head syndrome E. coli strains. No correlation was found between adhesion or haemagglutination patterns and pathogenicity. Only six of the 50 strains revealed invasive capacity and the strain that best invaded the cell lines was the one with the lowest LD50.     

猪致病性大肠杆菌耐药质粒检测及其中药消除作用研究

为探讨中药对河南省规模化养猪场致病性大肠杆菌(E.coli)耐药质粒消除的效果,本研究在对87个猪致病性E.coli分离株进行耐药质粒检测的基础上,将各分离株分别于含有不同浓度的石榴皮、黄芩、黄连等中药提取液的培养液中培养,进行耐药质粒消除试验;并采用影印培养法和纸片扩散法进行耐药性消除菌落筛选及其对抗菌药物敏感性恢复的检测.结果表明:各分离株均含有2个～8个质粒,大小约为1.0 kb～87.0 kb;经石榴皮、黄芩、黄连作用后,各分离株均可以获得数量不等的耐药性消除菌落,消除率为0.34％～16.2％,以黄连的平均消除率最高,为9.33％;在耐药性消除的各分离株中,有25个分离株未发生质粒消除,其余均消除了1条～3条质粒,并且对7种抗菌药物的平均敏感率由消除前的5.39％分别提高至60.4％、75.2％和76.5％.表明石榴皮、黄芩、黄连对猪致病性E.coli具有质粒消除和耐药性逆转作用,其中黄连作用效果最好.     

Spread of extended-spectrum beta-lactamase producing Escherichia coli isolates in Swedish broilers mediated by an incI plasmid carrying blaCTX-M-1

Background

The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli in European broiler populations is of concern due to the fact that third and fourth generation cephalosporins are deemed critically important in human medicine. In Sweden 34% of the broilers carry ESBL/pAmpC producing E. coli in their gut, despite the absence of a known selection pressure such as antimicrobial usages. The aim of the current study was to characterise a selection of E. coli strains carrying the bla_CTX-M-1, to determine if the spread was due to a specific clone.

Findings

Ten isolates carrying bla_CTX-M-1 from Swedish broilers belonged to eight different multi-locus sequence types with three isolates belonging to ST155. The ST155 isolates were identical as assessed by PFGE. The bla_CTX-M-1 was in all isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and sulfamethoxazole.

Conclusion

The occurrence of ESBL-producing E. coli in the Swedish broilers is not due to the emergence of a single clone, but rather the spread of a specific incI plasmid carrying bla_CTX-M-1.     

Identification of a new Escherichia coli She haemolysin homolog in avian E. coli

Haemolysin is one type of virulence factor that assists in the pathogenesis of Escherichia coli. Currently, hemolytic activity in E. coli has been attributed to haemolysin genes found in either uropathogenic or enterohemorrhagic E. coli. Both haemolysins are classified as RTX toxins because they both have repeats in toxin domains and share similar operon organization, sequence homology, and mechanisms of action. Haemolytic avian E. coli isolates, however, lack either E. coli haemolysin gene. To investigate the avian E. coli haemolysin, a genomic library was made from an avian pathogenic E. coli. A haemolytic clone that was isolated was shown to contain homology with sheA, an E. coli K- 12 gene which causes haemolysis when present in high copy number. The cloned haemolysin gene, hlyE, lacked the conserved amino acid sequence and accessory genes common to all RTX toxins. DNA hybridizations and polymerase chain reaction amplifications showed that the nucleotide sequences homologous to hlyE were not present in a collection of three O157: H7 E. coli, five haemolytic canine uropathogenic E. coli, one haemolytic O26 E. coli, and three haemolytic avian pathogenic E. coli. Thus we have identified a new E. coli haemolysin distinct from the RTX haemolysins and have shown that some avian pathogenic E. coli possess a haemolysin with no apparent homology to hlyE or RTX haemolysins.