Frequency of Escherichia coli strains producing the cytotoxic necrotizing factor (CNF1) in nosocomial urinary tract infections

The presence of cytotoxic necrotizing factor 1 (CNF1), together with various associated virulence factors (alpha-haemolysin, P-, S- and A-fimbriae), was screened in 175 uropathogenic Escherichia coli strains isolated from hospitalized adult patients. The cnf1 gene was detected in 30% of the selected strains independently of the severity of the clinical urinary infection. A significant association between CNF1, haemolytic activity and the products of the pap/sfa genes was found. However, CNF1 appeared not to play a major role in nosocomial E. coli urinary tract infections.     

Production of enterotoxin,verotoxin, hemolysin and cytotoxic necrotizing factor byEscherichia coli of intestinal and extraintestinal origin

Seventy-sixEscherichia coli strains were examined for heat-labile enterotoxin (LT), verotoxin (VT), hemolysin (HLy) and cytotoxic necrotizing factor (CNF). Thirty-six strains were isolated from patients suffering from diarrhea and forty from different extraintestinal infections. The number of LT-producing strains was low (2.6%) (one of intestinal and one of extraintestinal origin). Verotoxin was produced only by one extraintestinal strain. Four intestinal strains were hemolytic (11.2%) and also positive for CNF. From 24 hemolytic strains of extraintestinal origin (60%), 17 produced also CNF. Most of the hemolytic (30%) as well as CNF-producing strains (22.5%) were isolated from urine. Our results are similar to those of other studies confirming the close association between hemolysin and CNF production as well as a possible role of these toxic factors in pathogenesis of extraintestinal, infections caused byE. coli.     

Establishment of three categories of P-fimbriated Escherichia coli strains that show different toxic phenotypes and belong to particular O serogroups

Eight hundred and nineteen strains of Escherichia coli isolated in Spain between 1986 and 1991 from extraintestinal infections and feces of healthy controls were investigated for expression of P-fimbriae using a particle agglutination test. Among strains causing urinary tract infections, sepsis and other extraintestinal infections, P-fimbriae were found in 31% (130/420) (P < 0.001), 25% (30/118) (P < 0.001) and 12% (11/92) (P < 0.5) respectively. In contrast, only 7% (14/189) of faecal isolates from healthy individuals carried P-fimbriae. According to two more common toxic markers detected in this study (alpha-haemolysin and cytotoxic necrotizing factor type 1), P-fimbriated E. coli strains were grouped into three categories: haemolysin+cytotoxic necrosing factor+ (Hly+CNF1+) (68/185; 37%), haemolysin+cytotoxic necrosing factor- (Hly+CNF1-) (61/185; 33%) and Hly-CNF1- (56/185; 30%). The 185 P-fimbriated strains belonged to 17 different O serogroups. However, 148 (80%) were of one of six serogroups (O1, O2, O4, O6, O7 and O18). The most frequent serogroups determined in the Hly+CNF1+ strains were the O4 and O6 (53/68; 78%), in the Hly+CNF1- strains it was the O18 (27/61; 44%) and in the Hly-CNF1- strains the O1, O2 and O7 (41/56; 73%). The majority (160/185; 86%) of P-fimbriated E. coli expressed the mannose-resistant haemagglutinin type IVa.     

Pathogenic characteristics of Escherichia coli strains in cases of asymptomatic bacteriuria and symptomatic urinary tract infections

The aim of this study was to examine if E. coli isolated from asymptomatic bacteriuria differed in pathogenic features from strains isolated from symptomatic infections of urinary tract. In this study 130 strains of E. coli isolated from women having asymptomatic bacteriuria and 112 strains isolated from patients with symptoms of urinary tract infection were examined. It was shown that E. coli isolated from patients with symptomatic urinary tract infection showed the more frequently ability to cause mannose-resistant haemagglutination of human erythrocytes, resistance to bactericidal activity of serum and haemolytic properties than those isolated from asymptomatic bacteriuria. These strains showed also the higher ability to adhere to Vero cells in tissue culture. Among E. coli strains isolated from persons with asymptomatic bacteriuria the pathogenic features were most frequently found in strains from healthy women and the most rarely in isolated from diabetic women.     

Production of cytotoxin VT in enteropathogenic and non-enteropathogenic Eschericha coli strains of porcine origin

Abstract We have investigated the production of verotoxin (VT) in 55 enteropathogenic and 48 non-enteropathogenic Escherichia coli strains of porcine origin, belonging to 48 serotypes. VT cytotoxin was only produced by seven out of the eight enteropathogenic strains with serotypes O138:K81, O139:K82, O141:K85ab and O141:K85ac. Five haemolytic non-enteropathogenic E. coli , four with serotypes O75:K-, O75:K95 and O2:K2, and one not typable, synthesized a new thermolabile product not related with enterotoxin (LT) or VT, and which induced the formation of multinucleate cells in Vero monolayers.     

Incidence of toxigenic Escherichia coli in soft cheese made with raw or pasteurized milk

Soft cheeses made with raw (221 samples) or pasteurized (75) cow's milk were collected. Enterotoxigenic, verotoxigenic and necrotocigenic Escherichia coli strains were studied. Three raw milk cheeses were positive for toxigenic E. coli (1·4%) : the first with toxin CNF2 and serogroup O5 (40% of colonies studied with typical E. coli morphology) ; the second with VT and O2 (10%) ; and the third with LT and O51 (10%). Toxigenic E. coli of bovine origin can pass to the milk destined to make cheese, and survive. Soft cheese should be considered as a possible vehicle of infection in future investigations.     

Specificity of immunomodulator secretion in urinary samples in response to infection by alpha-hemolysin and CNF1 bearing uropathogenic Escherichia coli

Escherichia coli are the most common etiological agents of urinary tract infections (UTIs). Uropathogenic E. coli (UPECs) produce specific toxins including the cytotoxic necrotizing factor-1 (CNF1) and the alpha-hemolysin (alpha-Hly). CNF1 triggers, through Rho protein activation, a specific gene response of host cells, which results in the production for instance of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and the macrophage inflammatory protein-3alpha (MIP-3alpha). The alpha hemolysin alpha-Hly also triggers the production of inflammatory mediators. Cnf1 is always associated with alpha-hly in a pathogenicity island conserved among UPECs. Using two complementary approaches we have investigated whether alpha-hly and cnf1 bearing UPECs are associated with a specific type of UTI both in term of pathology and host response. Here we report that UPECs bearing alpha-hly/cnf1 have a prevalence of 50% in UPECs isolated from hemorrhagic UTIs, as compared to 30% in the overall UPEC population. In addition, we observed that MCP-1, and IL-8 to a lower extent, is produced in urine at higher concentrations in UTIs caused by UPECs carrying alpha-hly/cnf1.     

Cytotoxic and hemolytic activities in uropathogenic Escherichia coli

Fifty nine Escherichia coli strains obtained from patients with upper or lower urinary tract infections (UTI) and 30 E. coli strains isolated from stools of healthy individuals were tested for hemolytic and cytotoxic activities. Forty four percent of uropathogenic E. coli (UPEC) and 3.3% of fecal E. coli were hemolytic. Among the hemolytic UPEC, 92% produced alpha-hemolysin. A cytotoxic activity was detected in culture filtrates of 71% of UPEC strains and 30% of fecal E. coli. No relationship was found between cytotoxic and hemolytic activities or between cytotoxic titers and UPEC origin (upper or lower UTI). E. coli cytotoxin has a cytocidal activity against some epithelioid cultured cell lines (Vero, HeLa and Hep-2) but was almost inactive for avian-fibroblast cells. Cytotoxin-affected cells appeared rounded, refractile and detached from the surface of the vessel. Some characteristics exhibited by the cytotoxin as the morphological response induced on cells, the increasing of cytopathic effect with time, its irreversible cytocidal activity and its heat-lability resemble the properties described for E. coli Verotoxin (VT). Adherence to uroepithelial cells is recognized as a virulence factor for UPEC. It is suggested that cell damage by cytotoxic and adhering UPEC might contribute to E. coli virulence to urinary tract.     

Comparison of genomic profiles of Escherichia coli isolates from urinary tract infections

Formally included in the larger category of extraintestinal pathogenic Escherichia coli (ExPEC), the uropathogenic E. coli remains the most frequent cause of urinary tract infection (UTI), an important endemic health problem. The genomic DNA of E. coli urinary isolates from adults diagnosed with urinary tract infections and of E. coli fecal isolates from healthy subjects was analysed by PCR for the presence of virulence factor encoding genes pap, sfa/foc, afa, hly and cnf and by field inversion gel electrophoresis (FIGE) fingerprinting of XbaI DNA macrorestriction fragments. The aim was to obtain more detailed microbiological data regarding the community circulating strains in respect of their virulence potential and genetic relatedness. Almost 70% of the urinary strains carried at least one of the target virulence genes, and only 35.5% of the fecal E. coli strains were positive in the PCR screening. Taking into account the virulence genotypes exhibited, a part of the strains isolated from the urinary tract could be defined as belonging to the ExPEC pathotype. A unique FIGE profile was obtained for each of the selected isolates and the dendrogram generated by Taxotron software package analysis suggested a polyclonal population of potential uropathogenic strains clustered into 14 groups of only 60% similarity. For better understanding the epidemiology of UTIs, diseases commonly caused by such a heterogeneous species like E. coli, molecular analysis methods could be essential due to their increased power of identification and fingerprinting.     

泌尿系感染病原菌分布及耐药性分析

目的了解深圳市人民医院泌尿系感染病原菌的分布及耐药性,为临床医师合理使用抗菌药物提供科学依据。方法对655株泌尿系统感染病原菌进行常规鉴定,用k-B法或ATB-FUNGUS 3对其进行药敏试验。结果病原菌构成比前5位分别为大肠埃希菌（37.9%）、假丝酵母（18.0%）、肠球菌（13.1%）、肺炎克雷伯菌（6.6%）、铜绿假单胞菌（6.3%）。病原菌对各种抗菌药物产生了不同程度的耐药,肺炎克雷伯菌、铜绿假单胞菌对亚胺培南和美罗培南的耐药率为14.3%～26.8%。结论深圳市人民医院泌尿系感染病原菌主要以大肠埃希菌、假丝酵母和肠球菌等为主,病原菌对抗菌药物已产生了一定的耐药性,应加强监测与控制。     

Production of enterotoxins by strains of Escherichia coli isolated from pigs

The production of enterotoxins by 237 hemolytic strains of Escherichia coli isolated from pigs was determined with the use of CTE in CHO. Vero and Hela cells and ILT. More frequent (p less than 0.01) production of enterotoxins, determined by ILT, was found for the serotypes being pathogenic for the animals (63.8% of the strains). No correlation between intensity of ILT and particular serotype was observed. Both the serotypes pathogenic for pigs and other serotypes produced LT enterotoxins and ST toxin. The frequency of LT enterotoxin production was statistically insignificant compared to the frequency of ST enterotoxin production by strains with serotypes pathogenic for the pigs. Strains of E. coli producing only enterotoxin ST belonged both to the pathogenic serotypes as well as to other hemolytic serotypes. The cytotoxic activity of supernatants of E. coli strains with different serotypes isolated from pigs in Vero and Hela cells and simultaneous CTE in CHO cells was observed. This suggests the production by the strains of enterotoxin LT and cytotoxin VT. Seven out of the 96 isolates showing CTE in CHO cells gave no reaction in the ILT in pigs. This suggests the production by these isolates of a toxin (toxins) differing from the E. coli enterotoxins.     

Cloning of genes determining the production of vero cytotoxin by Escherichia coli

Sequences encoding the production of a cytotoxin (VT) active on Vero cells were cloned in Escherichia coli K12 from a VT-determining phage that originated in E. coli strain H19 of serotype O26.H11. Subcloning resulted in the identification of a 2.5 kb fragment that still coded for VT production. Mutagenesis with transposon Tn1000 was used to map VT sequences and a 0.75 kb probe was developed. In colony hybridization tests with strains isolated from patients with haemolytic uraemic syndrome or diarrhoea, this probe derived from the H19 VT genes detected only some of the VT+ strains belonging to serogroup 0157. A VT+ strain, E32511, serotype 0157.H-, which was negative in colony hybridization was the source of another VT-determining phage from which VT sequences were cloned. Southern hybridization of the VT genes from E32511 with the H19 probe was negative under stringent conditions but there was weak homology under conditions of low stringency. These results indicate that there are differences in the VT genes of pathogenic E. coli.     

绍兴地区2型糖尿病尿路感染病原菌及其耐药性分析

摘要：目的 回顾分析本院2型糖尿病伴尿路感染患者的病原菌分布特点及其耐药性,为指导临床用药提供参考。方法 收集2013年1月至2014年1月2型糖尿病合并尿路感染患者186例,留取中段尿分离培养病原菌,用VITEK-2细菌鉴定仪鉴定,纸片扩散法（K-B）测定药物敏感性。结果 186例患者中段尿共培养出病原菌137株,其中革兰阴性菌102株（74.45%）,革兰阳性菌21株（15.33%）,真菌14株（10.22%）。革兰阴性菌以大肠埃希菌为主,检出79株占57.66%,对青霉素类,头孢菌素类,喹诺酮类抗菌药耐药率较高均＞50.00%,对头霉素类药物如头孢替坦、含酶抑制剂复合物如哌拉西林/他唑巴坦以及碳青霉烯类药物敏感率均达100.00%;革兰阳性球菌以无乳链球菌为主,检出10株占7.30%,对克林霉素及红霉素耐药率较高,耐药率＞50.00%,而对氯霉素、呋喃妥因、利奈唑烷、替加环素、万古霉素敏感率均达100.00%。除1株光滑假丝酵母菌对氟康唑和伊曲康唑中介,其余13株真菌对两性霉素B、5-氟胞嘧啶、伏立康唑、氟康唑、伊曲康唑这5种抗真菌药物均敏感。结论 2型糖尿病患者发生尿路感染的病原菌以大肠埃希菌为主,且有较高的耐药率。     

Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases

A total of 78 E. coli strains isolated from adults with different types of urinary tract infections were screened by polymerase chain reaction for prevalence of genetic regions coding for virulence factors. The targeted genetic determinants were those coding for type 1 fimbriae ( fimH ), pili associated with pyelonephritis ( pap ), S and F1C fimbriae ( sfa and foc ), afimbrial adhesins ( afa ), hemolysin ( hly ), cytotoxic necrotizing factor ( cnf ), aerobactin ( aer ). Among the studied strains, the prevalence of genes coding for fimbrial adhesive systems was 86 %, 36%, and 23% for fimH, pap , and sfa/foc , respectively. The operons coding for Afa afimbrial adhesins were identified in 14% of strains. The hly and cnf genes coding for toxins were amplified in 23% and 13% of strains, respectively. A prevalence of 54% was found for the aer gene. The various combinations of detected genes were designated as virulence patterns. The strains isolated from the hospitalized patients displayed a greater number of virulence genes and a diversity of gene associations compared to the strains isolated from the ambulatory subjects. A rapid assessment of the bacterial pathogenicity characteristics may contribute to a better medical approach of the patients with urinary tract infections.     

Some biological features of Proteus bacilli. 2. Haemolytic activities of Proteus mirabilis and Proteus vulgaris strains

The haemolytic activities of Proteus mirabilis and P. vulgaris strains were studied under different conditions. No filterable alpha haemolysin could be detected in P. mirabilis uropathogens provided from patients with urinary tract infections. Together with the results presented in the accompanying paper, in which three clinical isolates with temporary ability to produce a soluble haemolysin were described, the occurrence of alpha haemolytic P. mirabilis isolates did not exceed 3%. Cell bound beta haemolysin is present in nearly 35% of P. mirabilis urinary strains. Another kind of haemolytic activity was observed when P. mirabilis and P. vulgaris strains were grown in liquid media supplemented with erythrocytes. During the logarithmic growth phase nearly 100% of P. mirabilis and P. vulgaris strains of various origin haemolyzed 100-50% of erythrocytes. Except for Serratia, the other representatives of Enterobacteriaceae did not demonstrate such activity in the same conditions. The preliminary characteristics of this phenomenon is given.     

traT gene sequences, serum resistance and pathogenicity-related factors in clinical isolates of Escherichia coli and other gram-negative bacteria

The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gram-negative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (50-70%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.     

Characteristic features of etiology and antibiotic susceptibility of urinary tract infection pathogens in patients with diabetes mellitus]

Patients with diabetes mellitus and urinary tract infection were examined. The structure of the pathogens and their antibiotic susceptibility were investigated. Escherichia coli was shown to be the main pathogen of urinary tract infections in the patients with diabetes mellitus. The highest activity against the E. coli isolates was revealed in amoxycillin/clavulanate (92% of the susceptible strains), the 3rd and 4th generation cephalosporins, carbapenems, amikacin (100%) and fluoroquinolones (96%). At the same time the isolates were resistant to aminopenicillins and co-trimoxazole (29.3 and 16% respectively).     

尿路感染相关病原菌的分布及耐药性分析

目的调查尿路感染病原菌的分布和耐药特点,为临床的抗感染治疗提供依据。方法收集2013年至2015年荆州市中心医院门诊和住院患者中,尿路感染患者送检的尿培养和血培养标本中检出的病原菌,采用Vitek2 Compact全自动微生物检测仪进行细菌鉴定,采用纸片扩散法和仪器法分别对革兰阴性杆菌和革兰阳性球菌进行药敏试验,药敏结果的判断依照CLSI M100-S24标准。数据分析采用WHONET5.6和SPSS 19.0软件,统计分析采用x~2检验。结果从尿路感染患者送检的标本中共检出各类非重复病原菌2 306株,其中门诊患者中检出19种100株,住院患者检出56种2 206株。导致尿路感染最多的两种病原菌为大肠埃希菌和粪肠球菌,分别检出1 241株和232株。导致尿脓毒血症最多的两种病原菌为大肠埃希菌和肺炎克雷伯菌,分别检出36株和10株。大肠埃希菌产ESBL.s率达67.9%,其对多种抗菌药物的耐药性均高于60.0%。粪肠球菌对大多数抗菌药物的耐药性均高于50.0%,仅对呋喃妥因和高浓度链霉素的耐药性较低,分别为12.0%和38.7%;未检出对万古霉素、利奈唑胺和替加环素耐药的粪肠球菌。结论导致尿路感染的病原菌种类繁多,大肠埃希菌和粪肠球菌是主要病原菌,其耐药情况严重;为保证治疗的有效性,临床医生应注重相关病原学和药敏检查结果。     

Role of verocytotoxigenic Escherichia coli in cattle and buffalo calf diarrhoea

Abstract 273 Strains of Escherichia coli isolated from diarrhoeic and healthy control cattle and buffalo calves in Sri Lanka, were tested for Verocytotoxin (VT) and for heat-stable (ST) and heat-labile (LT) enterotoxins. VT and ST toxigenic E. coli were significantly associated with diarrhoea, accounting for 28% and 18% of diarrhoeic episodes, respectively. LT toxigenic E. coli were not significantly associated with diarrhoea.     

Production of Vero cytotoxin by strains of Escherichia coli as related to the availability of iron

Abstract Vero cytotoxin (VT) producing strains of Escherichia coli (VTEC), including isolates from cases of haemolytic uraemic syndrome and infantile diarrhoea, were used to determine the effect of iron availability on the production of intra- and extracellular VT, with particular interest in elevating toxin production by low-level toxin producing VTEC. Culturing bacteria under iron restriction resulted in growth retardation and a decrease in the production of VT. For the routine detection of both high- and low-level VT-producing E. coli , there was no advantage to be gained by growing bacteria under iron restriction or using disrupted bacterial cell preparations; on the contrary, testing culture supernatants from bacteria grown in iron-replete media for approximately 14 h proved to be the most sensitive and accurate method for detecting VT and the resultant identification of VTEC.