Overlapped Sequence Types (STs) and Serogroups of Avian Pathogenic (APEC) and Human Extra-Intestinal Pathogenic (ExPEC) Escherichia coli Isolated in Brazil

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitD_chrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpV _Sakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some “zoonotic-related” STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.     

Complete Genome Sequence of the Novel Escherichia coli Phage phAPEC8

Bacteriophage phAPEC8 is an Escherichia coli-infecting myovirus, isolated on an avian pathogenic Escherichia coli (APEC) strain. APEC strains cause colibacillosis in poultry, resulting in high mortality levels and important economic losses. Genomic analysis of the 147,737-bp double-stranded DNA phAPEC8 genome revealed that 53% of the 269 encoded proteins are unique to this phage. Its closest relatives include the Salmonella phage PVP-SE1 and the coliphage rv5, with 19% and 18% similar proteins, respectively. As such, phAPEC8 represents a novel, phylogenetically distinct clade within the Myoviridae, with molecular properties suitable for phage therapy applications.     

禽致病性大肠杆菌江苏、安徽分离株的生物学特性分析

[目的]研究禽致病性大肠杆菌(Avian Pathogenic Escherichia coli,APEC)江苏、安徽分离株的优势血清型,并分析其生物学特性.[方法]对分离自病禽的细菌进行鉴定,采用玻片凝集法测定禽致病性大肠杆菌的血清型,PCR方法检测14种毒力基因的分布,采用美国临床和实验室标准化研究所的方法进行药物敏感性检测,改良结晶紫半定量法检测分离细菌的生物被膜形成能力. [结果]共分离到禽致病性大肠杆菌56株,血清型检测结果表明,O78血清型占64.29％,为主要血清型.毒力基因检测显示,fimC、pfs、ompA和luxS的阳性率超过90％.药物敏感性检测显示,58.93％的菌株对8种以上的药物耐受.生物被膜检测显示,有16株细菌生物被膜形成能力为中等以上,其中68.75％的菌株耐8种以上的药物.[结论]O78为主要流行的血清型.fimC、pfs、ompA和luxS基因为APEC保守基因.多重耐药性仍很普遍,细菌生物被膜与耐药性具有相关性.     

Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain

Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained bla _CTX-M-14, two bla _SHV-12, two bla _CMY-2 and one bla _SHV-2. Two strains harboured qnrA, and two qnrA together with aac(6’)-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured bla _CMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health.     

Complete Genome Sequence of the Novel Lytic Avian Pathogenic Coliphage NJ01

Bacteriophages of the C3 morphotype, characterized by very long heads that exceed their width several times, are extremely rare among the Podoviridae family members and constitute only 0.5% of over 5,500 phages that have been examined by the electron microscope (H. W. Ackermann, Arch. Virol. 152:227-243, 2007; H. W. Ackermann, Arch. Virol. 146:843-857, 2001). To date, among those phages proven to be C3, only coliphage phiEco32, Lactococcus phage KSY1, Vibrio phage 71A-6, and Salmonella enterica phage 7-11, but no avian pathogenic Escherichia coli (APEC) bacteriophages, have been completely sequenced (A. Chopin, H. Deveau, S. D. Ehrlich, S. Moineau, and M. C. Chopin, Virology 365:1-9, 2007; S. A. Khan, et al., Mol. Cell Probes 15:61-69, 2001; A. M. Kropinski, E. J. Lingohr, H. W. Ackermann, Arch. Virol. 156:149-151, 2011; D. Savalia, et al., J. Mol. Biol. 377:774-789, 2008) and are available in public databases. We isolated a bacteriophage from a scale duck market in Nanjing, Jiangsu province, named NJ01, that infects APEC. Sequence and morphological analyses revealed that phage NJ01 is a C3-like bacteriophage and belongs to the Podoviridae family. Here, we announce the complete genome sequence of phage NJ01 and submit the results of our analysis.     

基于RNA-Seq筛选APEC及其PhoP/Q缺失株感染雏鸡肠道免疫相关基因

本研究以禽致病性大肠杆菌(APEC)及其PhoP/Q缺失株感染雏鸡小肠为模型,以分析其免疫相关基因的表达变化为目的,采用转录组测序(RNA-Seq)技术对感染APEC及其PhoP/Q缺失株的雏鸡小肠样本RNA进行测序,分析免疫相关基因的表达变化,结果为野生株攻毒组与对照组相比、野生株攻毒组与缺失株攻毒组相比、缺失株攻毒组与对照组相比,分别筛选出131、105、172个差异表达基因(fold change≥2, FDR≤0.05),GO功能分类结果显示分别有87、99、159个基因得到注释,这些基因主要富集到氧化还原过程、脂蛋白转运、血管内皮细胞迁移、免疫反应、凋亡过程负调控、肝素结合、铁离子结合、CCR趋化因子受体结合等功能,得出APEC及其PhoP/Q缺失株感染雏鸡后引起机体肠道免疫相关基因变化的结论,根据GO功能注释筛选出PTPRC、LCP1、YFV等免疫相关基因,为深入研究雏鸡肠道免疫提供依据。     

致病性鸡大肠杆菌pilA基因和外膜蛋白C基因的克隆、表达及其免疫原性

根据GenBank公布的致病性鸡大肠杆菌的Ⅰ型菌毛pilA基因和外膜蛋白C基因序列,分别设计了两对引物,并以分离的致病性鸡大肠杆菌基因组为模板,经PCR特异性扩增出pilA基因和ompC基因,基因产物大小为别为549 bp和1104 bp,与GenBank报道的参考菌株的两个基因序列的同源性为高达98.18%和97.28%.将扩增得到的两个基因分别定向克隆到原核表达载体pET-28a中,得到两个重组质粒pETpilA和pETompC.转化大肠杆菌BL21(DE3)中,得到重组菌株BL21(pETpilA)和BL21(pETompC),经IPTG诱导后,SDS-PAGE分析分别可见表达的20 kD和40.9 kD的特异条带;Western blotting结果表明,两种蛋白可与抗体发生特异性结合,说明其具有良好的免疫原性.将表达的菌毛蛋白和外膜蛋白的菌株分别制成基因工程疫苗,免疫小鼠后,具有很好的保护能力.表明这两株基因工程菌株有望作为鸡致病性大肠杆菌基因工程疫苗的候选生产菌株.     

Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens.     

Characterization of a sequence (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli

Summary A sequence (hlyR) of about 600 bp which enhances the expression of hemolysin (HlyA) more than 50-fold was identified in the plasmid pHly152-specific hemolysin (hly) determinant. Deletion of this entire hlyR sequence led to the same low level of hemolysin synthesis and excretion as that expressed by the recombinant plasmid pANN202-312. HlyR was active in cis but its activity was orientation-dependent. The enhancing sequence, hlyR, is separated from the promoter phlyI transcribing hlyC, hlyA and possibly hlyB by more than 1.5 kb including an IS2 element. Stepwise removal of the hlyR sequence from its 5 end by exonuclease III (ExoIII) digestion yielded several types of deletion mutants which expressed decreasing amounts of hemolysin. A similar observation was made when hlyR was shortened by ExoIII from its 3 end, which suggests that more than one functional region may be present in the hlyR sequence. A deletion of 717 bp within the adjacent IS2 element reduced the activity of hlyR only slightly, indicating that IS2 is not directly involved in the enhancement mechanism but that it may support an optimal positioning in hlyR relative to the hly promoter. The nucleotide sequence of hlyR is rich in A+T and does not contain an extended open reading frame, but exhibits several sequence motives that may represent sites for protein binding and DNA bending.     

Sequence Diversity of Flagellin (fliC) Alleles in Pathogenic Escherichia coli

To study the molecular evolution of flagellin, the protein subunit specifying flagellar (H) antigens, the fliC genes from 15 pathogenic strains of Escherichia coli were amplified by PCR and sequenced. Comparison of fliC sequences of H6 and H7 strains revealed that alleles have a mosaic structure indicating the occurrence of past horizontal transfer of DNA segments between strains. The close similarity of H7 sequences also indicates the exchange of an entire fliC H7 allele between distant clonal lineages. In addition, the ratio of silent substitutions to amino acid replacements suggests that a short segment in the central region of fliC has been under positive selection in the divergence of H6 and H7 alleles. Phylogenetic analysis demonstrates that the fliC sequences of O157:H7 and O55:H7 serotypes are nearly identical and highly divergent from those of E. coli strains expressing H6 and H2 flagellar antigens. A nonmotile clone of sorbitol-fermenting O157 has rapidly accumulated multiple mutations in fliC, presumably as a result of the silencing of flagellin expression.     

Novel Pathways Revealed in Bursa of Fabricius Transcriptome in Response to Extraintestinal Pathogenic Escherichia coli (ExPEC) Infection

Extraintestinal pathogenic Escherichia coli (ExPEC) has major negative impacts on human and animal health. Recent research suggests food-borne links between human and animal ExPEC diseases with particular concern for poultry contaminated with avian pathogenic E. coli (APEC), the avian ExPEC. APEC is also a very important animal pathogen, causing colibacillosis, one of the world’s most widespread bacterial diseases of poultry. Previous studies showed marked atrophy and lymphocytes depletion in the bursa during APEC infection. Thus, a more comprehensive understanding of the avian bursa response to APEC infection will facilitate genetic selection for disease resistance. Four-week-old commercial male broiler chickens were infected with APEC O1 or given saline as a control. Bursas were collected at 1 and 5 days post-infection (dpi). Based on lesion scores of liver, pericardium and air sacs, infected birds were classified as having mild or severe pathology, representing resistant and susceptible phenotypes, respectively. Twenty-two individual bursa RNA libraries were sequenced, each yielding an average of 27 million single-end, 100-bp reads. There were 2469 novel genes in the total of 16,603 detected. Large numbers of significantly differentially expressed (DE) genes were detected when comparing susceptible and resistant birds at 5 dpi, susceptible and non-infected birds at 5 dpi, and susceptible birds at 5 dpi and 1 dpi. The DE genes were associated with signal transduction, the immune response, cell growth and cell death pathways. These data provide considerable insight into potential mechanisms of resistance to ExPEC infection, thus paving the way to develop strategies for ExPEC prevention and treatment, as well as enhancing innate resistance by genetic selection in animals.     

禽病原性大肠杆菌aes-31基因突变株的构建和致病性鉴定

【目的】通过禽病原性大肠杆菌(APEC)aes-31突变株的构建和动物实验来初步鉴定此基因片段对E058株的毒力影响。【方法】对在芯片杂交试验中筛选到的APEC E058株体外表达差异基因片段aes-31(来自SSH方法筛选到的E058株特异片段),用SSH引物从重组质粒中扩增出目的片段,克隆到pGEM-Teasy Vector中,然后用SphⅠ和SpeⅠ从中切下此片段,将之克隆到pMEG-375自杀性载体中,构建自杀性重组质粒pMEG375-aes-31,将突变载体转化到受体菌中,再和APEC E058株进行固相杂交,根据同源重组原理,筛选出基因突变株E058(Δaes-31)。【结果】E058株和突变株的LD50没有明显差别,突变株对35日龄SPF鸡的致死率高于E058株;两者接种鸡6 h内,E058(Δaes-31)突变株在各内脏器官和血液中的细菌数和E058株差异均不显著;接种鸡24 h后,E058(Δaes-31)突变株在脾脏和肺中的细菌数显著大于E058株,差异显著,E058(Δaes-31)突变株在心脏、肝脏和血液中细菌数显著大于E058株,差异极显著;接种鸡48 h后,E058(Δaes-31)突变株在心脏、肝脏和脾脏中的细菌数比E058株多,差异极显著,而肺脏和血液中的细菌数均无明显差异;48 h后突变株所引起的感染鸡的大肠杆菌病变比亲本株稍严重。【结论】以上数据表明aes-31有可能与E058株毒力的负调控有关。     

Characterization of the ves gene, which is expressed at a low temperature in Escherichia coli

A gene, designated ves, that is expressionally responsive to temperature was found in Escherichia coli. Experiments with a single-copy lacZ operon fusion and primer extension analysis revealed that ves was expressed at a low temperature with a peak around 25 degrees C but was hardly expressed at 42 degrees C. After a temperature downshift, the mRNA level increased until 6 to 12 h and then decreased. Consistently, an A + T-rich sequence similar to UP elements seen in cold-shock inducible cold-shock protein (Csp) genes was found up-stream of the ves promoter, and its 5'-untranslated region was found to share similarity with those of the cold-shock inducible and cold-adaptive cspA and cspB genes. Additionally, a putative down-stream box, which also exists in cold-inducible proteins, was found. The ves product was identified by overproduction and determination of its N-terminal sequence. Similarity of the C-terminal portion of Ves to the CspA family suggests that Ves belongs to this family. The results of gene-disruption experiments suggest that ves is not essential for E. coli.