Pathogenomics of uropathogenic Escherichia coli

Subset of faecal E. coli that can enter, colonize urinary tract and cause infection are known as uropathogenic E. coli (UPEC). UPEC strains act as opportunistic intracellular pathogens taking advantage of host susceptibility using a diverse array of virulence factors. Presence of specific virulence associated genes on genomic/pathogenicity islands and involvement of horizontal gene transfer appears to account for evolution and diversity of UPEC. Recent success in large-scale genome sequencing and comparative genomics has helped in unravelling UPEC pathogenomics. Here we review recent findings regarding virulence characteristics of UPEC and mechanisms involved in pathogenesis of urinary tract infection.     

Growth of uropathogenic Escherichia coli strains at solid surfaces

The adhesion and growth of two catheter-associated (O2K2 and O83K?) and two non catheter-associated (O111K58 and 0157K-) uropathogenic Escherichia coli strains on glass, poly(methyl methacrylate) (PMMA), a negatively charged copolymer of MMA and methacrylic acid (MAA) and a positively charged copolymer of MMA and trimethylaminoethyl methacrylate chloride (TMAEMA-Cl) were studied. The solid surfaces were placed in a parallel plate perfusion system. After preadhesion of the bacteria onto the surfaces, growth was initiated by perfusing the system with MacConkey broth. Growth was measured by counting adherent bacteria as a function of time. Bacterial strains were characterized by means of water contact angle, microbial adhesion to hydrocarbon (MATH), anion exchange resin retention (ARR) and zeta potential measurements. Solid surfaces were characterized by means of water contact angle and zeta potential measurements. The catheter-associated strains had significantly higher water contact angles, zeta potentials and ARR values than the non catheter-associated strains. Non catheter-associated strains did not grow at the surfaces used. Catheter-associated strains did not grow at the positively charged surface but exhibited growth at the other surfaces. Strains grew more rapidly at surfaces with a relatively high negative zeta potential and a low water contact angle than at surfaces with a relatively low negative zeta potential and a high water contact angle. The growth of strain O2K2 on glass was significantly reduced when urine instead of MacConkey broth was used as perfusion medium.     

The glycobiology of uropathogenic E. coli infection: the sweet and bitter role of sugars in urinary tract immunity

Urinary tract infections (UTI) are among the most prevalent infectious diseases and the most common cause of nosocomial infections, worldwide. Uropathogenic E. coli (UPEC) are responsible for approximately 80% of all UTI, which most commonly affect the bladder. UPEC colonize the urinary tract by ascension of the urethra, followed by cell invasion, and proliferation inside and outside urothelial cells, thereby causing symptomatic infections and quiescent intracellular reservoirs that may lead to recurrence. Sugars, or glycans, are key molecules for host–pathogen interactions, and UTI are no exception. Surface glycans regulate many of the events associated with UPEC adhesion and infection, as well as induction of the host immune response. While the bacterial protein FimH binds mannose‐containing host glycoproteins to initiate infection and UPEC‐secreted polysaccharides block immune mechanisms to favour intracellular replication, host glycans on the urothelial surface and on secreted glycoproteins prevent or limit infection by inhibiting UPEC adhesion. Given the importance of glycans during UTI, here we review the glycobiology of UPEC infection to highlight fundamental sugar‐mediated processes of immunological interest for their potential clinical applications. Interdisciplinary approaches incorporating glycomics and infection biology may help to develop novel non‐antibiotic‐based therapeutic strategies for bacterial infections as the spread of antimicrobial‐resistant uropathogens is currently threatening modern healthcare systems.     

Pathogenicity island markers in commensal and uropathogenic Escherichia coli isolates

Uropathogenic isolates of Escherichia coli (UPEC) contain blocks of DNA, termed pathogenicity islands (PAIs), that contribute to their virulence. Two multiplex PCR assays were developed to detect eight PAI markers among 50 commensal E. coli and 100 UPEC isolates. In total, 40% of commensal isolates and 93% of UPEC carried PAIs. Despite this difference, the distribution of various PAIs showed the same pattern in both groups, with the most prevalent being PAI IV(536) (38% commensal vs. 89% UPEC), followed by PAI I(CFT073) (26% vs. 73%), PAI II(CFT073) (14% vs. 46%), PAI II(J96) (8% vs. 34%), PAI I(536) (8% vs. 33%) and PAI II(536) (4% vs. 20%). PAI III(536) was detected only in UPEC (2%), while PAI I(J96) was not detected in any isolate. Although the mean number of PAIs per isolate was higher among UPEC (2.97) than in commensal (0.98) isolates, there were no statistical differences among group B2 E. coli from the two origins; however, commensal isolates from groups D and B1 appeared to be less virulent than pathogenic isolates. Regardless of their phylogenetic group, nearly all the commensal and UPEC isolates with the same number of PAIs had the same PAI combinations. Although group B2 E. coli are uncommon among commensal intestinal flora, they are highly virulent when present, suggesting that the intestinal flora may act as a reservoir for bacteria that can cause urinary tract infection.     

致肾盂肾炎大肠杆菌132的比较蛋白质组学研究

目的 研究致肾盂肾炎大肠杆菌(UPEC)132与UPEC J96及非致病性大肠杆菌K-12MG1655的蛋白质组表达差异.方法 采用双向凝胶电泳技术(2-DE)比较UPEC 132、UPEC J96与非致病性大肠杆菌K-12 MG1655的菌体蛋白表达图谱差异,差异蛋白用胰蛋白酶进行胶内酶切,肽混合物使用基质辅助激光解吸-电离飞行时间质谱仪(MALDI-TOF-MS)进行质谱分析,将肽质量指纹谱数据输入互联网上的蛋白质数据库进行检索.结果 UPEC 132识别蛋白点数(466±11)明显高于E.coli K-12 MG1655(338±15),也高于UPEC J96(382±12);3菌株共有的蛋白点为298个,2株UPEC共有的蛋白点为56个,UPEC 132特有的蛋白点为89个.MALDI-TOF-MS分析获得UPEC 132特异的及显著上调表达的蛋白点22个,涉及毒力因子、物质代谢转运、蛋白合成调节、生物氧化及未知功能的多种蛋白质.结论 UPEC菌株与非致病性大肠杆菌间的蛋白质组存在差异,UPEC 132与J96蛋白质组间也有显著不同,为其致病机制的深入研究提供线索.     

Identification of serotypes and virulence markers of Escherichia coli isolated from human stool and urine samples in Egypt

Purpose: Haemorrhagic colitis and haemolytic-uremic syndrome are associated with Shiga-toxin producing Escherichia coli (STEC). There are others DEC (Diarrhoeagenic E. coli) pathotypes responsible for outbreaks and others toxins associated to these. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include E. coli haemolysin (hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. Materials and Methods: In this study, the PCR assay was used to detect 12 E. coli genes associated with virulence (stx1, stx2, hylA, Flic h7 , stb, F41, K99, sta, F17, LT-I, LT-II and eaeA). Results: A total of 108 E. coli strains were serotyped into 64 typable strains. The investigated strains from the stool, 8/80 (10%) strains were O 164:K, while the 56/110 strains isolated from the urine were O126:K71 (44/110, 40%) and O 86:K 61 (12/110, 11%). The distribution pattern of the detected virulence genes was observed to be in the following order: F17 (10% from the stool and 44% from the urine), Sta (10% from the stool), hylA (10% from the stool and 44% from the urine), Stb (44% from the urine) and stx1 (27% from the urine). The 8 faecal strains encoded a combination of the F17, Sta and hylA genes, while the 56 urine strains encoded a combination of the F17 0+ Stb + hylA (44/110, 40%) and Stx1 only (12/60, 20%). Conclusion: This is the first report on the molecular characterization of E. coli diarrhoeagenic strains in Egypt and the first report on the potential role of E. coli in diarrhoea and urinary tract infections in a localized geographic area where the people engage in various occupational activities.     

E.coli临床分离株的基因组结构分型

目的：通过基因组结构分析对临床分离的Escherichia coli(E.coli)进行分型，并探讨分型与临床疾病的关系。方法：取临床不同疾病病人的痰、尿、血、分泌物等标本，分离E.coli。用I-Ceu Ⅰ酶切全基因组DNA，用脉冲场凝胶电泳分离DNA片段后，根据酶切图谱的异同进行分型。结果：从临床上分离的64株E.coli中，62株有7个I-Ceu Ⅰ酶切位点，2株有8个I-Ceu Ⅰ酶切位点。菌株间I-Ceu Ⅰ酶切图谱差异明显。这些菌株根据基因组结构的差异分为32个型，分型与疾病之间的对应关系分散。结论：临床分离的E.coli基因组结构存在多样性，其与临床疾病之间的关系有待进一步探讨。     

Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis

Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli (UPEC) to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium (K-SFM) with epidermal growth factor (EGF) and bovine pituitary extract (BPE). Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678-54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnf1 of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.     

致肾盂肾炎大肠杆菌对人肾盂上皮原代细胞黏附的实验研究

目的建立人肾盂上皮原代细胞的培养方法，用于致肾盂肾炎大肠杆菌（UPEC）黏附作用的研究。方法利用复合PCR方法检测UPEC132菌株毒力基因hly和cnf1。采集人肾脏手术标本，利用组织块法取肾盂正常上皮组织，使用添加EGF及BPE的角朊细胞无血清培养基（K-SFM）进行原代细胞培养，经HE染色和免疫细胞化学染色进行鉴定。鉴定符合要求的人肾盂上皮原代细胞用于UPEC黏附试验，分别于不同作用时间观察黏附后细胞形态学变化，并计算黏附率和黏附指数。结果此研究制备的人肾盂上皮原代细胞具备移行上皮细胞特征。带有毒力基因hly和cnf1以的UPEC132菌株作用于人肾盂上皮原代细胞，15min后细菌开始黏附，120min达到高峰，黏附率为74．4％，黏附指数为34．0。显微镜下观察细胞形变、着色不均，胞浆和胞核都有改变。结论人肾盂上皮原代细胞可用于UPEC的黏附试验，为其致病机理的深入研究奠定基础。     

Production by Escherichia coli isolates of siderophore and other virulence factors and their pathogenic role in a cutaneous infection model

Escherichia coli isolates from urinary tract infections (UTIs) (n = 124), extra-urinary sites (n = 37) and normal faecal samples (n = 51) were examined for the presence of virulence factors, including siderophores (aerobactin and enterobactin). The proportion of aerobactin producers was significantly higher in UTI (69.4%; p 0.001) and extra-urinary samples (70.3%; p 0.007) than in controls (41.2%), while the proportion of enterobactin producers was significantly lower in the UTI samples than in the controls (p 0.027). In a cutaneous infection model, aerobactin-positive E. coli showed more growth than non-aerobactin and non-enterobactin isolates, even when other virulence factors were identical.     

Transurethral instillation with fusion protein MrpH.FimH induces protective innate immune responses against uropathogenic Escherichia coli and Proteus mirabilis

Urinary tract infections (UTIs) are among the most common infections in human. Innate immunity recognizes pathogen‐associated molecular patterns (PAMPs) by Toll‐like receptors (TLRs) to activate responses against pathogens. Recently, we demonstrated that MrpH.FimH fusion protein consisting of MrpH from Proteus mirabilis and FimH from Uropathogenic Escherichia coli (UPEC) results in the higher immunogenicity and protection, as compared with FimH and MrpH alone. In this study, we evaluated the innate immunity and adjuvant properties induced by fusion MrpH.FimH through in vitro and in vivo methods. FimH and MrpH.FimH were able to induce significantly higher IL‐8 and IL‐6 responses than untreated or MrpH alone in cell lines tested. The neutrophil count was significantly higher in the fusion group than other groups. After 6 h, IL‐8 and IL‐6 production reached a peak, with a significant decline at 24 h post‐instillation in both bladder and kidney tissues. Mice instilled with the fusion and challenged with UPEC or P. mirabilis showed a significant decrease in the number of bacteria in bladder and kidney compared to control mice. The results of these studies demonstrate that the use of recombinant fusion protein encoding TLR‐4 ligand represents an effective vaccination strategy that does not require the use of a commercial adjuvant. Furthermore, MrpH.FimH was presented as a promising vaccine candidate against UTIs caused by UPEC and P. mirabilis.     

IroN functions as a siderophore receptor and is a urovirulence factor in an extraintestinal pathogenic isolate of Escherichia coli

IroN was recently identified in the extracellular pathogenic Escherichia coli strain CP9. In this study experimental evidence demonstrating that IroN mediates utilization of the siderophore enterobactin was obtained, thereby establishing IroN as a catecholate siderophore receptor. In a mouse model of ascending urinary tract infection the presence of iroN contributed significantly to CP9's ability to colonize the mouse bladder, kidneys, and urine, evidence that IroN is a urovirulence factor. However, growth in human urine ex vivo and adherence to uroepithelial cells in vitro were equivalent for an isogenic mutant deficient in IroN (CP82) and its wild-type parent (CP9). Taken together, these findings establish that IroN is a siderophore receptor and a urovirulence factor. However, uncertainty exists as to the mechanism(s) via which IroN contributes to urovirulence.     

Uropathogenic Escherichia coli causes fibrotic remodelling of the epididymis

Despite antibiotic treatment, up to 40% of patients have impaired fertility after epididymitis due to serovars of Escherichia coli, a frequent pathogen. The reasons for infertility are unclear, but it may result from epididymal duct obstruction. To determine whether E. coli infection of the epididymis causes obstruction due to fibrosis, and to identify the key mediators, tissues from patients with epididymitis were assessed. Additionally, epididymitis was induced with uropathogenic E. coli (UPEC) or commensal serovars in wild‐type and MyD88^?/? mice, which are relatively unresponsive to bacterial pathogens. Epididymal organ cultures were treated with activin A and bacteria and their histology and levels of cytokines and fibrosis markers were analysed. Patients with epididymitis showed severe fibrosis of the epididymal duct. In mice, UPEC infection also caused fibrosis and ductal obstruction in the cauda epididymis. Levels of mRNA for fibrotic markers (α‐smooth muscle actin, fibronectin) and cytokines (activin A, TNFα, IL‐1α, IL‐1β, IL‐6) and total collagen levels were significantly elevated. This fibrotic response was blunted by the loss of MyD88. Activin A induced fibrosis in cultured epididymis, which was inhibited by the activin‐binding protein follistatin. In summary, bacterial epididymitis causes fibrosis and obstruction. The milder tissue damage in Myd88^?/? UPEC epididymitis highlights the importance of the host response to infection in causing epididymal damage. Elevated levels of activin A in vivo and fibrotic remodelling elicited by activin A in vitro indicate that this cytokine is a potential target for supplementary treatment to antibiotic therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

致肾盂肾炎大肠杆菌papA基因的克隆及序列分析

目的 对致肾盂肾炎大肠杆菌（ＵＰＥＣ）１３２株的ｐａｐＡ基因进行克隆和序列分析。方法 根据Ｆ１３型ｐａｐＡ基因序列设计引物,并在二引物５′端加上限制性内切酶ＥｃｏＲⅠ和ＢａｎＨⅠ的酶切位点。采用此引物扩增１３２菌株ｐ菌毛粘附基因群的ｐａｐＡ基因,扩增产物克隆人质粒,选取阳性重组质粒进行核苷酸序列分析。结果 ＵＰＥＣ１３２株经ＰＣＲ法扩增获得约７００ｂｐ的ｐａｐＡ片段,经克隆获得阳性重组质粒ｐＣＴ２     

Distribution of virulence determinants among antimicrobial-resistant and antimicrobial-susceptible Escherichia coli implicated in urinary tract infections

Introduction: Uropathogenic Escherichia coli (UPEC) rely on the correlation of virulence expression with antimicrobial resistance to persist and cause severe urinary tract infections (UTIs). Objectives: We assessed the virulence pattern and prevalence among UPEC strains susceptible and resistant to multiple antimicrobial classes. Methods: A total of 174 non-duplicate UPEC strains from patients with clinically significant UTIs were analysed for susceptibility to aminoglycoside, antifolate, cephalosporin, nitrofuran and quinolone antibiotics for the production of extended-spectrum β-lactamases and for the presence of six virulence determinants encoding adhesins (afimbrial, Type 1 fimbriae, P and S-fimbriae) and toxins (cytotoxic necrotising factor and haemolysin). Results: Relatively high resistance rates to nalidixic acid, ciprofloxacin, cephalothin and trimethoprim-sulfamethoxazole (82%, 78%, 62% and 59%, respectively) were observed. Fourteen distinct patterns were identified for the virulence determinants such as afaBC, cnfI, fimH, hylA, papEF and sfaDE. The toxin gene, cnfI (75.3%), was the second most prevalent marker to the adhesin, fimH (97.1%). The significant association of sfaDE/hylA (P < 0.01) among antimicrobial resistant and susceptible strains was also observed notwithstanding an overall greater occurrence of virulence factors among the latter. Conclusions: This study provides a snapshot of UPEC complexity in Jamaica and highlights the significant clonal heterogeneity among strains. Such outcomes emphasise the need for evidence-based strategies in the effective management and control of UTIs.     

大肠杆菌O120 O-抗原基因簇的破译及特异分子标识的鉴定

目的完成产志贺毒素大肠杆菌O120 OO-抗原基因簇的破译，筛选和鉴定检测用特异分子标识。方法利用鸟枪法进行O-抗原基因簇序列的测定，进行生物信息学方法分析发现基因并预测基因功能，利用PCR方法筛选针对大肠杆菌O120 O特异基因和特异引物，利用基因芯片方法筛选特异探针。结果O-抗原基因簇序列全长12485bp，共含有10个基因：dTDP-鼠李糖合成途径基因（rmlB、rmlD、rmlA和rmlC），O-抗原转运酶基因（uzx），O-抗原聚合酶基因（wzy），3个糖基转移酶基因和1个功能未确定的基因。另外筛选和鉴定了2个特异基因、4对特异引物和6条特异探针，在模拟样品的鉴定中得到验证。结论多种特异分子标识可从样品中特异地检测出大肠杆菌O120 O，具有快速、灵敏和准确进行分子分型的优点。     

Adhesins and invasins of pathogenic Escherichia coli

Pathogenic E. coli cause both intestinal and extra-intestinal infections in humans and animals. Bacteria must be able to adhere to host cells if they are to colonize and to invade their hosts. Numerous E. coli adhesins with different morphological features and receptor specificities have been identified. Many bacteria produce several adhesins with different receptor specificities. Although not all adhesin receptors have been identified yet, it appears that adhesins generally behave as lectins, recognizing oligosaccharide residues of glycoproteins or glycolipids. This review summarizes recent advances concerning host tissue colonization properties, providing new insights into adhesive organelle biogenesis in pathogenic E. coli and into the development of reservoirs of pathogenic bacteria in the host. To limit the length of this review, I will use examples of structural characteristics and invasive properties of a few bacterial adherence factors: type 1 pili, Afa adhesive sheath and some outer membrane adhesins.     

Detection of diarrheagenic Escherichia coli by multiplex PCR

Background: Diarrheagenic E.coli (DEC) are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus) forEnteropathogenic E.coli (EPEC), hlyA for Enterohemorrhagic E.coli (EHEC), elt and stla for Enterotoxigenic E.coli (ETEC), CVD432 for Enteroaggregative E.coli (EAEC) and ial for Enteroinvasive E.coli (EIEC). Results: In 200 children with diarrhea 52 (26%) DEC infections were found. Among 100 controls 8 (8%) DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%)and controls (5,5%), followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5%) and 3 (1.5%) of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.     

Detection of a plasmid-encoded pathogenicity island in F18+ enterotoxigenic and verotoxigenic Escherichia coli from weaned pigs

Most virulence genes of enterotoxigenic Escherichia coli (ETEC) are located on plasmids. The gene for heat-stable enterotoxin I (sta) is part of the transposon Tn1681, and the heat-stable enterotoxin II (stb) gene was described to be part of the transposon Tn4521. In the studies presented here, we describe the linkage of the sta and stb genes on an approximately 10-kb fragment designated as toxin-specific locus (TSL). The TSL has been isolated from the 120-kb virulence plasmid pTC of the porcine ETEC strain 2173 that produces F18 fimbriae. The nucleotide sequence of the TSL fragment was determined. Sequences in the flanking regions of the sta gene indicated the presence of Tn1681, but--unexpectedly--flanking sequences of the neighbouring stb gene were completely different from those of Tn4521. The 10-kb TSL is part of a 40-kb fragment that contains the replication origin of pTC. This 40-kb fragment was mobilised into plasmid pACYC177 and the nucleotide sequence of the bordering fragments was determined. The 40-kb fragment was flanked by IS10 elements at both ends, indicating the existence of a new 40-kb pathogenicity island (PAI) in strain 2173. Several F4(K88)+ ETEC and F18+ ETEC as well as F18+ E. coli strains producing enterotoxins and verotoxin-2 (ETEC/VTEC) from weaned pigs of different geographical origin were tested for the flanking regions by PCR to see if they belong to the "Tn4521-like" or the "pTC-like" stb type. It turned out that the Tn4521-like stb-type was characteristic of F4(K88)+ ETEC, while the pTC-like stb type was present in most F18+ ETEC and F18+ ETEC/VTEC, although polymorphism was observed both in the K88 and F18 groups. These results suggest the existence and worldwide spread of a new plasmid-encoded pathogenicity island in porcine post weaning ETEC and ETEC/VTEC strains producing F18 fimbriae.