Effect of fur mutation on acid-tolerance response and in vivo virulence of avian septicemic Escherichia coli

The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria. In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined. The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain. However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation. These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E. coli, as detected by the ability to cause septicemia in our experimental infection.     

抑制差减杂交筛选禽致病性大肠杆菌基因组差异片段及其分析

采用抑制差减杂交技术(Suppression subtractive hybridization,SSH)对禽致病性大肠杆菌E037株(血清型O78)与非致病菌株K-12MG1655以及同一O2血清型高致病菌株E058与低致病菌株E526进行基因组差异片段克隆与分析。从E037株中共检出17个特异性差异片段,E058株中共检出32个特异性差异片段。经同源分析,这些序列可分为4类:质粒相关序列、噬菌体相关序列、已知功能序列、未知功能序列。这些差异片段包含许多重要的大肠杆菌毒力相关基因,如大肠杆菌素、气杆菌素受体、铁基因簇等。49个片段中,14个片段与其它微生物基因组同源性较高。结果表明,大肠杆菌高致病株与低致病菌株或非致病菌株基因组间存在较多差异基因,其中包括毒力、毒力相关基因、代谢以及噬菌体等基因成分。     

禽源大肠杆菌的分离及其毒力因子的检测

从临床疑似大肠杆菌感染的病禽组织中分离到69株细菌(其中鹅源29株,鸡源40株);通过常规形态学、培养特性和生化特征的研究,确定为大肠杆菌。PCR检测表明,其中46株(66.7%)为F1 大肠杆菌,10株(14.5%)为F1 HPI 大肠杆菌,2株(2.9%)为HPI 大肠杆菌;通过比较还发现,F1菌毛和HPI在鹅源和鸡源大肠杆菌中以及不同脏器来源的菌株中具有相似的分子流行病学。O抗原鉴定结果表明鹅源大肠杆菌的O抗原型主要有O26、O78、O18、O117,鸡源大肠杆菌的O抗原型主要有O109、O24、O18、O139、O78。药敏试验表明,其中绝大多数菌株对先锋霉素V、呋喃妥因、庆大霉素敏感,对环丙沙星因菌株差异而不同,林可霉素、四环素、多粘菌素多不敏感。     

New Role for the ibeA Gene in H2O2 Stress Resistance of Escherichia coli

ibeA is a virulence factor found in some extraintestinal pathogenic Escherichia coli (ExPEC) strains from the B2 phylogenetic group and particularly in newborn meningitic and avian pathogenic strains. It was shown to be involved in the invasion process of the newborn meningitic strain RS218. In a previous work, we showed that in the avian pathogenic E. coli (APEC) strain BEN2908, isolated from a colibacillosis case, ibeA was rather involved in adhesion to eukaryotic cells by modulating type 1 fimbria synthesis (M. A. Cortes et al., Infect. Immun. 76:4129-4136, 2008). In this study, we demonstrate a new role for ibeA in oxidative stress resistance. We showed that an ibeA mutant of E. coli BEN2908 was more sensitive than its wild-type counterpart to H(2)O(2) killing. This phenotype was also observed in a mutant deleted for the whole GimA genomic region carrying ibeA and might be linked to alterations in the expression of a subset of genes involved in the oxidative stress response. We also showed that RpoS expression was not altered by the ibeA deletion. Moreover, the transfer of an ibeA-expressing plasmid into an E. coli K-12 strain, expressing or not expressing type 1 fimbriae, rendered it more resistant to an H(2)O(2) challenge. Altogether, these results show that ibeA by itself is able to confer increased H(2)O(2) resistance to E. coli. This feature could partly explain the role played by ibeA in the virulence of pathogenic strains.     

Investigation of shiga toxin-producing Escherichia coli in avian species in India

AIMS: To investigate the presence or absence of shiga toxin-producing Escherichia coli (STEC) in avian species in India. METHODS AND RESULTS: Faecal samples originating from 500 chicken and 25 free flying pigeons were screened for the presence of E. coli. A total of 426 (chicken, 401; pigeons, 25) E. coli strains were isolated. Of 426 E. coli strains, 387 were grouped into 77 serogroups, while 70 and 59 strains were untypable and rough, respectively. All isolates were subjected to multiplex polymerase chain reaction (m-PCR) for the detection of stx(1), stx(2), eaeA, hlyA and saa genes. None of the E. coli strains studied showed the presence of stx(1), stx(2) or their variants and saa genes. Overall 11 (2.74%) and seven (1.74%) strains from chickens possessed eaeA and hlyA genes, respectively, while as only six (1.49%) strains from chickens possessed both eaeA and hlyA genes. O9, O8, O60 and O25 serogroups were most predominant of which there were 24 (5.63%), 23 (5.39%), 23 (5.39%) and 20 (4.69%) strains, respectively. None of the isolates from pigeons showed the presence of any of the virulence genes studied. CONCLUSIONS: STEC are absent in chickens and pigeons. However, further studies are required in this direction to confirm or contradict our findings. E. coli strains originating from birds are carrying a low percentage eaeA or hlyA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first attempt to investigate STEC in chickens and free flying pigeons in India. The chickens and pigeons cannot be considered as important carrier of STEC in India.     

Colonization of chicken cecae by Escherichia coli associated with hemorrhagic colitis.

Bacterial enumeration, histologic examination, and immunoperoxidase staining demonstrated the ability of an Escherichia coli strain associated with hemorrhagic colitis (serotype O157:H7) to colonize chicken cecae for up to 90 days postinoculation after a peroral challenge at 1 day of age. The bacteria induced mild, transient, mucous membrane damage confined to the proximal cecae of healthy, normal-appearing chickens, principally at 14 to 28 days postinoculation. Attachment, effacement, and penetration of the cecal surface epithelium by E. coli O157:H7 were observed. With the exception of splenic, hepatic, and cecal tonsil immune-related changes and cecal damage and colonization, no other organ systems or portions of the gastrointestinal tract were affected by the bacteria. Bacterial counts indicated that E. coli O157:H7 was predominantly present in the cecae (often at levels greater than 10(6) CFU/g of tissue and contents) and to a lesser extent in the colon. Our results suggest that E. coli O157:H7 colonizes chicken cecae and is passed through the colon with fecal excrement. The ability of this organism to colonize chicken cecae indicates that chickens may serve as hosts and possibly as reservoirs for E. coli O157:H7.     

Pathogenicity of pap-negative avian Escherichia coli isolated from septicaemic lesions

Recent studies by DNA-DNA hybridisation assays conducted on a large collection of Escherichia coli strains isolated from chickens, ducks and turkeys suffering from colibacillosis, showed that 76% of the strains were negative for the presence of the pap gene cluster. The objective of this paper was to study the virulence associated with the avian E. coli strains negative for the P fimbriae, but carrying the f17 or the afa-8 gene cluster coding for adhesins associated with strains pathogenic for mammals. Three strains carrying the f17 fimbriae and three carrying the afa-8 adhesin-encoding gene cluster were studied in three in vivo experimental models of avian colibacillosis: subcutaneous inoculation of 1-day-old chicks, inoculation of specific-pathogen-free (SPF) chickens via the intra-thoracic air sac, and intra-tracheal inoculation of axenic chickens. The results showed that the six P-negative E. coli isolates carrying the f17 or the afa-8 gene cluster were lethal for 1-day-old chicks. They were also able to reproduce clinical signs and lesions of colibacillosis (aerosacculitis, pericarditis, perihepathitis), with bacteraemia and septicaemia, in SPF chickens inoculated via the thoracic air sacs as well as in axenic chickens inoculated by the intra-tracheal route. Further studies with f17 and afa-8 allelic mutants constructed by disruption must be performed to confirm a role of F17 fimbrial and Afa-VIII afimbrial adhesins in the pathogenesis of avian colibacillosis.     

Avian air sac and plasma proteins that bind surface polysaccharides of Escherichia coli O2

Some serovars of Escherichia coli, mainly O2 and O78, are responsible for air sac and systemic infections in farm-raised turkeys (Meleagris gallopavo) and chickens (Gallus gallus). We looked in air sac surface fluid from young turkeys to identify proteins that bind surface polysaccharides of pathogenic respiratory E. coli O2. Turkey air sac surface fluid was subjected to affinity chromatography on Toyopearl AF-Epoxy-650M, coupled with either lipopolysaccharide (LPS) or lipid-free polysaccharide (LFP) purified from an avian pathogenic E. coli O2 isolate. A multimeric protein termed lipid-free polysaccharide binding protein-40 (LFPBP-40) composed of six covalently associated subunits of approximately 40 kDa was isolated by elution from LFP by EDTA or L-rhamnose. An analogous protein in air sac fluid proteins bound to intact E. coli O2 and eluted with L-rhamnose or N-acetylglucosamine (GlcNAc). The N-terminal amino acid sequence of LFPBP-40 DINGGGATLPQHLYLTPDV was related to the N-terminus of fragment 3 of a partially characterized human protein possessing T cell stimulation activity in synovial membrane of rheumatoid arthritis patients. However, endogenous amino acid sequences were unrelated to other known proteins. LFPBP-40 was immunoreactively distinct from pulmonary collectins and ficolins. These studies demonstrate a novel avian respiratory soluble lectin that can bind surface polysaccharides of pathogenic E. coli responsible for respiratory disease.     

应用DNA芯片技术对禽致病性大肠杆菌可能致病基因体外表达水平的研究

应用DNA芯片研究禽致病性大肠杆菌可能致病基因的表达.构建禽致病性大肠杆菌毒力基因、潜在毒力基因的DNA芯片,应用基因芯片技术对同属O2血清型的禽高致病性大肠杆菌E058株和低致病性大肠杆菌E526株在体外LB培养基和鸡血清培养状态下进行差异表达分析.结果:在体外LB静置培养状态下,低致病株E526与高致病株E058相比共有16个差异基因,均为下调基因.在鸡血清静置培养中,E526与E058相比共有15个差异基因,均为下调基因.应用基因芯片成功筛选了禽致病性大肠杆菌在体外不同条件下的毒力基因及可能毒力基因中差异表达基因,表明一些铁摄取系统相关基因对APEC的毒力较重要,同时也筛选出了一些新的可能致病基因aes-1,aes-2,aes-3,aes-4,aes-6,aes-8,aes-10,aes-13,aes-15,aes-31等.     

Prevention of drug-resistant Escherichia coli colonization in chickens by treatment with a faecal fluid

This study was performed to prove that intestinal colonization in chickens by resistant Escherichia coli strains present in the environment might be prevented when faeces in which sensitive E. coli strains were dominant was administered to newly hatched chicks. The appearance of resistant E. coli strains was markedly reduced. Escherichia coli O49:H12 was the sensitive E. coli strain which formed the major colonizer in the intestinal tract. In young chickens, this strain persisted as a major component, and even when it was a minor colonizer in the faecal fluid administered, it appeared as a major component soon afterwards. This strain is considered to be a good colonizer in the gut of young chickens.     

Role of rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator sigma(S) and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10(4) CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P 相似文献   

The genome sequence of avian pathogenic Escherichia coli strain O1:K1:H7 shares strong similarities with human extraintestinal pathogenic E. coli genomes

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include human uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC). Regardless of host of origin, ExPEC strains share many traits. It has been suggested that these commonalities may enable APEC to cause disease in humans. Here, we begin to test the hypothesis that certain APEC strains possess potential to cause human urinary tract infection through virulence genotyping of 1,000 APEC and UPEC strains, generation of the first complete genomic sequence of an APEC (APEC O1:K1:H7) strain, and comparison of this genome to all available human ExPEC genomic sequences. The genomes of APEC O1 and three human UPEC strains were found to be remarkably similar, with only 4.5% of APEC O1's genome not found in other sequenced ExPEC genomes. Also, use of multilocus sequence typing showed that some of the sequenced human ExPEC strains were more like APEC O1 than other human ExPEC strains. This work provides evidence that at least some human and avian ExPEC strains are highly similar to one another, and it supports the possibility that a food-borne link between some APEC and UPEC strains exists. Future studies are necessary to assess the ability of APEC to overcome the hurdles necessary for such a food-borne transmission, and epidemiological studies are required to confirm that such a phenomenon actually occurs.     

The ets sequence is required for induction of erythroblastosis in chickens by avian retrovirus E26.

E26 is a replication-defective avian retrovirus that causes an erythroblastic leukemia in vivo and transforms hematopoietic precursor cells of both the erythroid and the myeloid lineages in vitro. The E26 genome contains two sets of cell-derived sequences, ets and myb. myb sequences are also present in avian myeloblastosis virus, which transforms myeloblasts exclusively. To determine whether the ets sequence is responsible for the erythroid specificity of E26, we analyzed the transforming activities of several viruses carrying mutations in the ets sequence constructed in vitro. The mutant viruses retained the ability to transform myeloid cells in vitro, indicating that the myb oncogene is sufficient for this viral function. However, the ets-deficient viruses did not cause an overt leukemia in chickens. The results indicate that the ets sequence is required for the induction of erythroblastosis by E26.     

禽致病性大肠杆菌（CVCC1565）中耶尔森菌强毒力岛核心区的检测

采用PCR法,检测了大肠杆菌（CVCC1565）中耶尔森菌强毒力岛（high pathogenicity island,HPI）核心区的irp1、irp2、irp3、irp4、irp5及fyuA基因片段,并与小肠结肠炎耶尔森菌毒力岛的类似基因进行同源性比较。结果显示,E.coliCVCC1565菌株irp1、irp2、irp3、irp4、irp5及fyuA基因大小分别为799bp、414bp、798bp、504bp、758bp、948bp,与GenBank中公布的小肠结肠炎耶尔森菌（Yersinia enterocoliticaO：8 WA）HPI的irp1、irp2、irp3、irp4、irp5及fyuA基因同源性分别达到98%、98%、98%、95%、98%、98%。研究结果表明禽致病性大肠杆菌标准株（CVCC1565）携带耶尔森菌强毒力岛基因,这几个毒力岛基因在小肠结肠炎耶尔森菌和禽致病性大肠杆菌之间可能存在水平性转移。     

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.     

禽病原性大肠杆菌iro和tsh基因缺失株的构建

通过SSH和SCOTS研究, 铁系统(Iro)和温度敏感性血凝素(Tsh)在禽病原性大肠杆菌(APEC)的感染中可能发挥重要作用。基因检测发现, 在243个禽源大肠杆菌分离株中, 有205株为iro+菌株, 其中高、中度和低致病株分别为89.8%(184/205)、8.8%(18/205)和1.5%(3/205); 有167株为tsh+菌株, 高、中度、低致病株分别为87.4%(146/167)、12.6%(21/167)和0%(0/167), 结果显示iro+或tsh+株大多数为高致病株。为了确定iro和tsh基因在APEC致病力中的作用, 以APEC E037株为基础, 通过自杀性载体分别构建了iro和tsh基因缺失突变株E037(Δiro)、E037(Δtsh)和E037(ΔiroΔtsh)。动物感染性试验表明, 突变株在鸡体内的繁殖能力和致病性均明显下降, 但两个基因的协同致病作用不显著。进一步证实Iro和Tsh为APEC重要的致病因子。     

Prevention of drug-resistant Escherichia coli colonization in chickens by treatment with a faecal fluid

This study was performed to prove that intestinal colonization in chickens by resistant Escherichia coli strains present in the environment might be prevented when faeces in which sensitive E. coli strains were dominant was administered to newly hatched chicks. The appearance of resistant E. coli strains was markedly reduced. Escherichia coli O49:H12 was the sensitive E. coli strain which formed the major colonizer in the intestinal tract. In young chickens, this strain persisted as a major component, and even when it was a minor colonizer in the faecal fluid administered, it appeared as a major component soon afterwards. This strain is considered to be a good colonizer in the gut of young chickens.     

Infections with Avian Pathogenic and Fecal Escherichia coli Strains Display Similar Lung Histopathology and Macrophage Apoptosis

The purpose of this study was to compare histopathological changes in the lungs of chickens infected with avian pathogenic (APEC) and avian fecal (A(fecal)) Escherichia coli strains, and to analyze how the interaction of the bacteria with avian macrophages relates to the outcome of the infection. Chickens were infected intratracheally with three APEC strains, MT78, IMT5155, and UEL17, and one non-pathogenic A(fecal) strain, IMT5104. The pathogenicity of the strains was assessed by isolating bacteria from lungs, kidneys, and spleens at 24 h post-infection (p.i.). Lungs were examined for histopathological changes at 12, 18, and 24 h p.i. Serial lung sections were stained with hematoxylin and eosin (HE), terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) for detection of apoptotic cells, and an anti-O2 antibody for detection of MT78 and IMT5155. UEL17 and IMT5104 did not cause systemic infections and the extents of lung colonization were two orders of magnitude lower than for the septicemic strains MT78 and IMT5155, yet all four strains caused the same extent of inflammation in the lungs. The inflammation was localized; there were some congested areas next to unaffected areas. Only the inflamed regions became labeled with anti-O2 antibody. TUNEL labeling revealed the presence of apoptotic cells at 12 h p.i in the inflamed regions only, and before any necrotic foci could be seen. The TUNEL-positive cells were very likely dying heterophils, as evidenced by the purulent inflammation. Some of the dying cells observed in avian lungs in situ may also be macrophages, since all four avian E. coli induced caspase 3/7 activation in monolayers of HD11 avian macrophages. In summary, both pathogenic and non-pathogenic fecal strains of avian E. coli produce focal infections in the avian lung, and these are accompanied by inflammation and cell death in the infected areas.