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.     

Serotypes and virulence genes of extraintestinal pathogenic Escherichia coli isolates from diseased pigs in China

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates were detected in 315/3127 (10.1%) diseased pigs from 19 provinces of China; the frequency of isolation increased from 3.1% in 2004 to 14.6% in 2007. All isolates were characterised for O serogroups, haemolysis, phenotypic and genotypic antimicrobial resistance, virulence genes and pathogenicity. The most prevalent serogroups were O161, O8, O11, O138, O101 and O26; 83/315 (26.3%) isolates were haemolytic. Forty percent of isolates in phylogenetic groups B2 and D were highly virulent porcine ExPEC strains. Thirty-three putative extraintestinal virulence factor genes that are normally associated with human and/or avian ExPEC strains were widely present in porcine isolates. These results indicate that ExPEC are prevalent in pigs in China and represent a potential public health threat.     

Relationship between serotype, virulence and SDS-PAGE protein patterns of Haemophilus parasuis

141 Haemophilus (H.) parasuis and 8 H. parasuis-like strains from different farms were serotyped according to Morozumi and Nicolet (1986 b) as well as to Bakos et al. (1952). It was possible to classify 72.8% of the investigated strains. 7 out of 12 serotypes have been described for the first time. The high specificity in the agar gel precipitation test was not reproducible in the more sensitive dot-blot procedure. The dot-blot results point to a participation of non-immunogenic polysaccharides in the detection reaction. The serotypes SV 1, SV 5, SV Jena 6 and SV Jena 10 proved to be highly virulent in SPF pigs, SV 2 and SV 4 were of medium virulence. The other serotypes were found to be nonvirulent. Unencapsulated strains and isolates of serotype SV 5 prevailed in animals with Glasser's disease. 23 H. parasuis and 3 H. parasuis-like strains were examined in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of protein profiles of whole-cell lysates, 23 of them could be assigned to 5 groups. Apart from the highly virulent strains of serovar 1, which belonged to PAGE type III, all other highly virulent strains of the serovars SV 5, SV Jena 6 and SV Jena 10 were grouped into PAGE type I. No correlation could be found between PAGE type on the one hand and virulence or origin of isolates on the other hand.     

TonB is essential for virulence in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) strains have multiple iron-uptake systems that facilitate adaptation to iron-restricted environments and are believed to assist in colonisation of the host. These systems include several TonB-dependent transporters of ferri-siderophores encoded by the chromosome and the large virulence plasmid common to APECs. The tonB gene of the virulent APEC strain E956 was replaced with a selectable antibiotic resistance marker using Lambda Red recombinase mutagenesis. The phenotype of the ΔtonB E956 mutant was compared to the parent strain under various culture conditions and in chickens experimentally infected via the respiratory route. The mutant was resistant to streptonigrin, impaired in its ability to adapt to growth in iron-depleted medium and had greater tolerance of oxidative stress than the parental strain. The mutant was avirulent in chickens, did not affect the growth of chicks and colonisation was mostly limited to the trachea. This study has demonstrated that TonB is essential for virulence in APEC.     

Biological and immunological characterization of pili of Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry

Pili of Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry were isolated and purified by sucrose-density-gradient centrifugation. Each serotype expressed only one type of pilus. The pili of the three serotypes had similar densities and were morphologically similar by electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, however, showed that they were slightly different in subunit molecular mass. Slide agglutination, immunodiffusion, and immunoblot tests were used to test for antigenic relationships between these pili and reference pili. Pili of serotype O78 were type 1, but pili of serotypes O1 and O2 were not, as once believed. However, pili of serotype O2 reacted positively with anti-type 1 serum in immunoblot assay, suggesting the presence of some common antigenic epitopes among these pili.     

Antimicrobial resistance of Escherichia coli O26 and O111 isolates from cattle and their characteristics

The present study was to investigate antimicrobial resistance profiles of Escherichia coli O26 and O111 from cattle and to characterize the virulence genes of the resistant isolates. This paper reports the high prevalence of antimicrobial resistant E. coli O26 and O111 from cattle. Among 37 E. coli O26 and 25 E. coli O111 isolates from the fecal specimens obtained from cattle, 26 (70%) and 15 (60%) were resistant to at least one antibiotic, respectively. Forty (98%) of the 41 resistant isolates were resistant to two or more antibiotics. Among the 22 antibiotics tested in this study, ampicillin was the most common antibiotic that the isolates were resistant to, followed by tetracycline and streptomycin. None of the isolates were resistant to fluoroquinolones, such as ciprofloxacin, ofloxacin and norfloxacin, and to ceftriaxone, amikacin and imipenem. Eighteen different resistant types among the 41 isolates were observed by the cluster analysis. The most frequent antibiotic-resistance type was ampicillin-tetracycline-streptomycin-cephalothin-sulfisoxazole-ticarcillin-kanamycin-minocycline-piperacillin-chloramphenicol, which accounted for 9 (22%) of the resistant isolates. The observation of frequent and multiple resistances to antibiotics highlights the need for their careful use if their benefits are to be preserved. PCR analysis of the EHEC virulence markers showed that 25 of the resistant E. coli O26 and O111 isolates tested positive for stx2 or both stx1 and stx2. This suggests that the majority of these isolates can cause serious diseases in humans and may complicate the future therapeutic options under development.     

Antimicrobial resistance, virulence profiles, and phylogenetic groups of fecal Escherichia coli isolates: a comparative analysis between dogs and their owners in Japan

In this study, fecal Escherichia coli isolates (n=188) from 34 dog-owner pairs and 26 healthy control humans (2 isolates per individual) were tested for susceptibility to 6 antimicrobials and screened for virulence genes. Genetic diversity between canine and owner isolates was evaluated by pulsed-field gel electrophoresis (PFGE). Canine isolates exhibited significantly different rates of resistance to four and two antimicrobials, compared to control and owner isolates, respectively. Of the genes examined, the prevalence of sfa, hly, and cnf genes in canine isolates were higher than in control isolates, but not than in owner isolates. These results suggest that characteristics of owner isolates are somewhat similar to canine isolates, compared to isolates from non-dog owners. In addition, PFGE analysis revealed that transfer of E. coli between owners and their dogs had occurred within 3/34 (8.8%) households. Considering the effects of dog ownership on the population of E. coli isolates from owners, further epidemiological studies are required.     

Virulence factors of Escherichia coli, with emphasis on avian pathogenic isolates

E. coli bacteria isolated from localized and systemic disease processes in poultry are designated as Avian Pathogenic E. coli (APEC). The disease-inducing potential of these isolates has been explained by the occurrence of specific virulence factors. Despite the extensive literature on virulence factors for E. coli, unambiguous markers of virulence have not been identified yet. The relationship between serotyping and virulence is not straightforward either and raises the question whether E. coli infections in poultry should mainly be considered as opportunistic. Investigations into the occurrence of certain (combinations of) virulence factors in APEC isolates as virulence markers should fulfil the molecular version of Koch's postulates if the former question is to be answered.     

Transposon mutagenesis used to study the role of complement resistance in the virulence of an avian Escherichia coli isolate.

The role of complement resistance in the virulence of an avian Escherichia coli isolate was examined with transposon mutagenesis. A suicide plasmid containing a kanamycin-encoding mini-transposon was used to transform a virulent complement-resistant avian E. coli isolate. A less resistant mutant was identified that contained a transposon insertion in a plasmid and in the chromosome. This loss of complement resistance was associated with a drop in virulence in an embryo assay. No other phenotypic changes were detected in the mutant. These results suggest that complement resistance is associated with the virulence of this organism.     

Antimicrobial susceptibility and molecular characterization of avian pathogenic Escherichia coli isolates

Ninety-five avian pathogenic Escherichia coli (APEC) isolates recovered from diagnosed cases of avian colibacillosis from North Georgia between 1996 and 2000 were serotyped and examined for typical virulence-factors, susceptibility to antimicrobials of human and veterinary significance, and genetic relatedness. Twenty different serotypes were identified, with O78 being the most common (12%). The majority of the avian E. coli isolates (60%), however, were non-typeable with standard O antisera. Eighty-four percent of isolates were PCR positive for the temperature-sensitive hemagglutinin (tsh) gene and 86% positive for the increased serum survival (iss) gene. Multiple antimicrobial-resistant phenotypes (> or =3 antimicrobials) were observed in 92% of E. coli isolates, with the majority of isolates displaying resistance to sulfamethoxazole (93%), tetracycline (87%), streptomycin (86%), gentamicin (69%), and nalidixic acid (59%). Fifty-six E. coli isolates displaying resistance to nalidixic acid were co-resistant to difloxacin (57%), enrofloxacin (16%), gatifloxacin (2%), and levofloxacin (2%). DNA sequencing revealed point mutations in gyrA (Ser83-Leu, Asp87-Tyr, Asp87-Gly, Asp87-Ala), gyrB (Glu466-Asp, Asp426-Thr), and parC (Ser80-Ile, Ser80-Arg). No mutations were observed in parE. Twelve of the quinolone-resistant E. coli isolates were tolerant to cyclohexane, a marker for upregulation of the acrAB multi-drug resistance efflux pump. Quinolone-resistant isolates were further genetically characterized via ribotyping. Twenty-two distinct ribogroups were identified, with 61% of isolates clustering into four major ribogroups, indicating that quinolone resistance has emerged among multiple avian pathogenic E. coli serogroups and chromosomal backgrounds.     

肠道外致病性大肠杆菌外膜蛋白研究进展

肠道外致病性大肠杆菌病(Extraintestinal pathogenic Escherichia coli,ExPEC)是一种常发肠道外疾病,危害人类、动物的健康,也导致禽类产蛋量的下降及肉用动物的上市延迟,严重的则引起死亡。因此,对于肠道外致病性大肠杆菌的致病机制也是许多学者研究的热点,肠道外致病性大肠杆菌致病机理的研究近些年主要集中在脂多糖(LPS)、外膜蛋白(Outer membrane protein,Omp)、菌毛、鞭毛、等毒力因子方面。     

Characterization of a series of transconjugant mutants of an avian pathogenic Escherichia coli isolate for resistance to serum complement

Colibacillosis, caused by avian pathogenic Escherichia coli (APEC) is a major problem for the poultry industry resulting in significant losses annually. Previous work in our lab and by others has shown that the increased serum survival gene (iss) is a common trait associated with the virulence of APEC. This gene was first described for its contributions to E. coli serum resistance. However, recently published research has called the contribution of iss to this trait into question. In the present study, the level of serum resistance conferred on an E. coli isolate by iss is examined. Additionally, the contribution of lambda bor gene to E. coli serum resistance is studied, as iss is thought to be derived from bor and bor occurs commonly among E. coli. To better understand the iss and bor contributions to serum resistance, a series of iss and bor mutants was generated. An iss deletion (iss-) mutant showed a significant drop in its resistance to serum. Similarly, a bor mutant showed a drop in serum resistance but not as drastic as that observed with the iss mutant, suggesting that iss contributes more to serum resistance than bor in this E. coli strain. Also, when iss was reintroduced into the iss- mutant the wild-type level of serum resistance was restored, confirming that the deletion of iss was responsible for the change in resistance seen in the mutant.     

Antigenic relatedness and partial amino acid sequences of pili of Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry

Pilus proteins from Escherichia coli serotypes O1, O2, and O78 pathogenic to poultry were compared with regard to their antigenic relatedness and partial amino acid sequences. Agglutination, immunodiffusion, and immunoblot assays with polyclonal antibodies to these pili showed that these pili not only share some common antigens but also contain antigens unique to each pilus. The partial amino-terminal amino acid sequences support our earlier findings that the pili are different but contain some structural homologies.     

Antimicrobial susceptibilities, serogroups, and molecular characterization of avian pathogenic Escherichia coli isolates in Japan

In total, 83 avian pathogenic Escherichia coli (APEC) isolates from avian colibacillosis during a period from 2001 to 2006 in Japan were investigated for serogroups, typical virulence factors, antimicrobial susceptibility, and genetic relatedness. The most common serogroup was O78 (30.1%); 80.7% of isolates harbored the iss gene and 55.4% of isolates harbored the tsh gene. Antimicrobial resistance of the isolates was found for ampicillin (77.1%), oxytetracycline (75.9%), kanamycin (36.1%), fradiomycin (33.7%), trimethoprim (25.3%), enrofloxacin (21.7%), and florfenicol (6.0%). Although multiple antimicrobial-resistant phenotypes (three or more antimicrobials) accounted for 54.2% of isolates, no isolate exhibited resistance to all agents tested. The fluoroquinolone-resistant isolates had point mutations in GyrA (Ser83 --> Leu, Asp87 --> Asn) and ParC (Ser80 --> Ile, Glu84 --> Gly). Of 18 enrofloxacin-resistant E. coli isolates, nine isolates belonged to serotype O78. In PFGE analysis, eight of the nine enrofloxacin-resistant O78 isolates were classified into an identical cluster. This suggests that a specific genotype of fluoroquinolone-resistant O78 APEC may be widely distributed in Japan.     

鸭源大肠杆菌分离鉴定及其毒力基因检测

从信阳市部分规模化鸭场疑似大肠杆菌感染病料中,通过传统分离培养及phoA基因检测,分离得到19株大肠杆菌。对分离菌进行种系发育分群、动物致病性试验和毒力基因检测。系统发育分子分群结果显示,19株分离菌中10株(52.63%)属于B2群,7株(36.84%)属于D群,1株(5.26%)属于B1群,1株(5.26%)属于A群。动物致病性试验结果显示,高致病性菌株9株(47.38%),中致病性菌株7株(36.84%),低致病性菌株3株(15.78%)。毒力基因的检测结果显示,ompT、iroN、hlyF、iutA和Iss五种毒力基因的检出率均为89.47%;fimC、marA和irp2基因检出率为63.16%;fyuA基因检出率为42.11%。     

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.     

广东地区5种优势血清型猪大肠杆菌的外膜蛋白型研究

对从广东地区部分猪场分离的大肠杆菌进行血清型鉴定后,选取优势血清型O₉、O₂₆、O₆₀、O₁₀₁和O₁₀₇的8个致病性较强的分离株,采用超声波裂解、N-十二烷基肌氨酸钠处理和超速离心技术提取大肠杆菌的外膜蛋白（OMP）,利用SDS-PAGE电泳进行OMP分型。结果8个菌株共有3个OMP型（Ⅰ～Ⅲ型）,其中O₂₆、O₁₀₁、O₁₀₇型的3个分离株的外膜蛋白型由1条带组成,为OMP-Ⅰ型;O₆₀、O₁₀₁、O₁₀₇型的4个分离株的外膜蛋白型由2条带组成,为OMP-Ⅱ型;O₉型的1个分离株的外膜蛋白型由3条带组成,为OMP-Ⅲ型。这表明,优势血清型O₉、O₂₆、O₆₀、O₁₀₁、O₁₀₇的分离株OMP型不单一;同一血清型的分离株,存在不同OMP型;而不同血清型的分离株却可有相同的OMP型。     

Occurrence and virulence patterns of E. coli O26, O103, O111 and O145 in slaughter cattle

The study attempted to investigate the occurrence of non-O157 E. coli serogroups O26, O103, O111 and O145 in cattle at slaughter and to determine the virulence potential of these isolates. A total of 399 fecal samples were analyzed by selective plating and E. coli isolates were characterized by polymerase chain reaction (PCR) for the genes vtx1, vtx2, eae and EHEC hlyA. Immunomagnetic separation (IMS) is required to increase the efficiency of the isolation procedure. E. coli O26, O103, O111 and O145 were recovered from 24 (6%) fecal samples. E. coli O26 and O103 seemed to be more abundant in slaughter cattle than E. coli O111 and O145. Sixteen out of the 24 isolates harbored vtx genes. All vtx-positive isolates harbored one or more additional virulence factors. Six out of the 8 vtx-negative isolates harbored eae and/or EHEC hlyA, whereas 2 strains harbored none of the tested virulence genes.     

致狐狸肺炎大肠杆菌血清鉴定与毒力基因检测

为了确定狐狸肺炎源致病性大肠杆菌(Escherichia coli,E.coli)流行血清型与毒力基因的携带情况并分析其相关性,2016—2017年采集河北省部分地区患肺炎死亡的狐狸的肺脏、心血等组织142份中分离到了105株E.coli,且RT-PCR检测犬瘟热病毒为阴性,人工感染小鼠致病性试验结果显示105株分离菌株中77株为致病性E.coli。采用玻片凝集试验和PCR方法分别检测77株致病性E.coli血清型和毒力基因,结果显示:77株致病性E.coli有9种血清型,其中以O1、O8、O78、O12为流行的优势致病血清型,77株致病性E.coli携带14种不同毒力基因,以irp2、fyuA、papC、fimC、iucD^a、colv、hlyF、ompT 8种毒力基因检出率较高,在32.5%～98.7%之间,其余毒力基因检出率较低。狐狸肺炎源致病性E.coli的优势血清型携带的毒力基因较多,至少携带9种毒力基因,流行的优势血清型与毒力基因之间具有一定的相关性。狐狸肺炎源E.coli流行血清型代表株TS(O1)1、QH(O8)2、QH(O78)3、TS(O12)4均对狐狸很强的致病性。本试验为狐狸肺炎源致病性E.coli防控提供了参考依据。