Virulence factors associated with Escherichia coli present in a commercially produced competitive exclusion product

In this study, we assessed the pathogenic potential of Escherichia coli associated with a commercial competitive exclusion (CE) product by examining the phenotypic characteristics associated with E. coli virulent for humans and domestic animals. Most E. coli isolates were capable of proliferating in iron-deplete chicken sera. Interestingly, none of the E. coli isolates from the commercial CE product contained the bacterial adhesin Tsh characteristic of avian pathogenic E. coli associated with airsacculitis and colisepticemia. In terms of virulence potential for humans, most E. coli isolates (78%) were sensitive to killing by 12.5% human sera. Because of their sensitivity to human sera, the E. coli in the CE product are not likely to cause a serious systemic infection in humans and, therefore, do not present a risk of causing septicemia in humans. Because these isolates also lack the gene tsh, they are also less likely to cause systemic disease or airsacculitis in poultry than pathogenic strains commonly isolated from diseased birds.     

Virulence genes in bla(CTX-M) Escherichia coli isolates from chickens and humans

The aim of this study was to determine the presence of virulence genes in isolates of CTX-M Escherichia coli from diseased chickens, from healthy chickens and from urinary tract infections in people. Three CTX-M E. coli strains from three different instances of disease in poultry (two of which were E. coli related) were tested for bla(CTX-M) sequence type and replicon type. Additionally, they were tested for the presence of 56 virulence genes (encoding fimbriae, adhesins, toxins, microcins and iron acquisition genes) using a micro-array. Results were compared to the virulence genes present in isolates from 26 healthy chickens and from 10 people with urinary tract infections. All genes found in isolates from diseased birds, including the astA (heat stable toxin) and tsh (temperature sensitive haemagglutinin) genes which have previously been associated with colibacillosis in chickens, were also present in isolates from healthy birds. However, 6/10 of the virulence genes found were exclusive to isolates from humans. Genes exclusive to chicken isolates included ireA (sidephore receptor), lpfA (long polar fimbriae), mchF (microcin transporter protein) and tsh whilst genes exclusive to human isolates included ctdB (cytolethal distending toxin), nfaE (non-fimbrial adhesion), senB (plasmid encoded enterotoxin) and toxB (toxin B). The results support previous findings that CTX-M E. coli strains in chickens are generally different from those causing disease in humans, but genes such as astA and tsh in isolates from diseased birds with colisepticaemia were also present in isolates from healthy birds.     

Virulence factors of Escherichia cofi from cellulitis or colisepticemia lesions in chickens

This study was designed to compare virulence factors of cellulitis-derived Escherichia coli to colisepticemic E. coli in order to clarify whether E. coli associated with cellulitis comprise a unique subset of pathogenic E. coli. Isolates were tested for serotype, capsule, aerobactin production, colicin production, the presence of the iss gene, and serum resistance. Untypable isolates made up the greatest percentage of each group. Serotypes O2 and O78 were the most commonly identified among both groups of isolates. No statistical differences in the distribution of aerobactin or colicin production, capsule, or iss gene were observed between groups. Cluster analysis showed that 90% of the E. coli isolates had greater than 42% livability in serum-resistance tests. No separation of colisepticemic vs. cellulitis E. coli isolates was observed on the basis of SR. Colicin production by E. coli was highly correlated with serum resistance (P = 0.0029). These data suggest that cellulitis E. coli have virulence traits similar to those of colisepticemic E. coli.     

Production of H2S by Escherichia coli isolated from poultry: an unusual character useful for epidemiology of colisepticemia

Eleven isolates of H2S-producing Escherichia coli were recovered from necropsy materials of chickens with symptoms and lesions of colisepticemia on Saudi Arabian broiler farms. Results of 19 out of 20 biochemical reactions studied were typical for E. coli. Hydrogen sulfide production by the E. coli isolates was used as an epidemiological marker to pinpoint a breeding farm as the probable source of these strains, which were then transferred to progeny farms, where colisepticemia occurred. This finding was confirmed by the presence of the same antigenic structure (O78:H-) and by the same drug-resistance pattern (a multiple resistance to streptomycin, sulfathiazole, and tetracycline) in the isolates.     

Virulence factors of avian pathogenic Escherichia coli strains isolated from chickens with colisepticemia in Japan

The virulence factors of avian pathogenic Escherichia coli (APEC) isolated in Japan were investigated. Serogroups O, serotypes K1 and K5, and genes cva C, iss, iutA, papA, tsh, and usp, which have been thought to be related to virulence, were examined for their association with E. coli strains isolated from diseased and healthy chickens. The frequently recognized serogroups O1, O2, and O78 were found in 56 of 125 (44.8%) strains of diseased chickens (APEC) versus 13 of 100 (13.0%) strains of healthy chickens (commensal E. coli), a significant difference at risk ratio < 0.01. Although iss, iutA, and tsh were widely distributed in the APEC irrespective of O serogroup, papA, usp, and the K1 serotype were detected in serogroup O2 of APEC. The kfiD gene related to the K5 capsule and VT, LT, and ST genes related to exotoxins were not detected in any strains examined.     

Virulence factors of avian Escherichia coli

A total of 45 strains of Escherichia coli isolates from chickens with colisepticemia were examined for virulence factors commonly found in pathogenic groups of E. coli. These strains were studied for the following: pathogenicity in 1-day-old chicks; toxin, hemolysin, and colicin production; cell invasiveness and adherence; hemagglutination for fimbriae detection; serum resistance; aerobactin production in iron-limited conditions; and plasmid content. The characteristics exhibited by virulent strains were invasion for HeLa and chicken fibroblast cells, serum resistance, colicin V, and aerobactin production. None of the isolates were toxigenic or positive in hemagglutination tests. The molecular genetic studies of the virulence factors by agarose electrophoresis showed that the plasmids of these strains are of high molecular weight.     

Comparison of chicken plasma and guinea pig serum in a quantitative microtiter method of determining microbial complement resistance.

A quantitative microtiter method using chicken plasma is described for determining the degree of complement resistance or sensitivity of avian Escherichia coli isolates. Results obtained with the microtiter method using chicken plasma were compred with results obtained using commercially available standardized guinea pig serum as the source of complement. The test organisms consisted of five isolates of E. coli isolated from chickens. Three isolates were from flocks with colisepticemia; one was from a flock with omphalitis; and one isolate was a non-pathogenic control. Data were accumulated from the five avian E. coli isolates incubated at 35 C with either chicken plasma or guinea pig serum and with heat-inactivated chicken plasma or guinea pig serum. The microtiter results of the chicken plasma and guinea pig serum had a statistically positive correlation. The use of commercially available guinea pig serum in the test system will allow for standardization of this method.     

105株携带耶尔森菌强毒力岛大肠杆菌的毒力基因型和部分菌株毒力因子序列研究

为了解105株携带耶尔森菌强毒力岛(HPI)的大肠杆菌(E.coli)中相关毒力因子的流行情况和基因序列,根据GenBank中参考序列设计引物,采用PCR方法对广东地区养殖场来源的105分离株HPI+E.coli的fyuA、tsh、iucD、iss 4种毒力基因进行检测,统计基因类型;并对部分分离株的5种毒力基因(irp2和fuA、tsh、iucD、iss)进行了克隆与序列分析.结果显示105株HPI+E coli中4种毒力因子携带情况不尽一致,基因fyuA、tsh、iucD和iss的阳性率分别为55.24％、17.14％、49.52％和23.81％,105株HPI+E.coli共有13种基因型;分析表明,除iss基因与参考序列的同源性在88.0 ％～90.9％外,irp2、fyuA、tsh、iucD4种基因与GenBank中参考序列的同源性高达96％以上;广东省养殖场E.coli毒力因子基因型复杂,并以基因型irp2+ fyuA+ iucD+和仅含irp2+的菌株分离率最高,分别为17.14％和28％.     

Distribution of lipopolysaccharide core types among avian pathogenic Escherichia coli in relation to the major phylogenetic groups

Five distinct lipopolysaccharide (LPS) core types, namely R1-R4 and K12 have been identified in Escherichia coli. The aims of this study were to determine, primarily by means of PCR, the distribution of those oligosaccharide core types among avian pathogenic E. coli and their relationship to phylogenetic groups. To identify putative avian pathogenic E. coli, serum resistance and the presence of three virulence genes encoding temperature sensitive haemagglutinin (tsh), increased serum survival (iss) and colicin V (cvaC) were determined. Of the 143 clinical isolates examined 62% possessed the R1 core, 22% were R3, 13% were R4 and 3% were R2. Fifty commensal isolates consisted of 58% with R1 core, 38% with R3 core, 4% with R4 core, and none with R2. None of the isolates were of K12 core type. The distribution of core oligosaccharide types in clinical and commensal isolates were not statistically significant (P=0.51). Three genes, tsh, iss and cvaC were found in E. coli of all four core types. The genes tsh (P<0.001) and iss (P=0.03412) were significantly associated with the R4 core oligosaccharide type. The isolates containing R4 core type LPS were mainly confined to phylogenetic group D. The widespread R1 core type showed less ability to possess virulence genes and 83% were in the phylogenetic group A. Results of this study indicated that E. coli with R1, R2, R3 and R4 were important in causing infections in chickens and further, the E. coli with R4 core type were less common among commensals, possessed more virulence genes and were related to phylogenetic groups pathogenic for poultry.     

Characterization of Escherichia coli strains obtained from layer chickens affected with colibacillosis in a commercial egg-producing farm

The present study reports colibacillosis of layer chickens in a commercial egg-producing farm in western Japan. Three flocks of chicken at 18-21 weeks of age were affected during the initiation of egg lay. Postmortem examination revealed pericarditis, perihepatitis, airsacculitis, subcutaneous inguinal lesion, and injured cloaca. Escherichia coli was isolated from the lesions of the affected birds. Twenty-two of 26 E. coli isolates (84.6%) obtained from 18 birds in the 3 flocks showed pulsed-field gel electrophoresis (PFGE) patterns that were considered to be closely associated to each other and arbitrarily designated as pattern A. All the 22 isolates with the PFGE pattern A harbored the putative virulence genes, astA, iss, iucD, tsh, and cva/cvi. Additional 2 PFGE patterns (B and C) were also found in E. coli isolates obtained from the affected flocks and had the putative virulence genes in combinations different from those in the pattern A strains. The results suggested that certain E. coli virulence genes and host factors, such as initiation of egg lay may be associated with occurrence of colibacillosis.     

Congo red medium to distinguish between invasive and non-invasive Escherichia coli pathogenic for poultry

In the course of our molecular studies of virulence factors associated with invasive avian Escherichia coli infections, it was first necessary to distinguish between common E. coli and those that cause septicemia in poultry. We found a direct correlation between the ability of clinical isolates of E. coli to bind Congo red dye (CR) and their ability to cause septicemic infection in chickens. This finding was supported by bacteriological studies of 30 broiler flocks (26 sick and 4 healthy) and by virulence studies in chickens and mice. All 144 isolates of E. coli from internal tissues of diseased birds were determined to be CR-positive (red colonies). Congo-red-positive E. coli colonies were isolated from air sacs, pericardium, liver, lung, joint fluid, and heart blood of chickens with lesions of colisepticemia. In contrast, of 170 E. coli isolates from the poultry house environment and from the trachea and cloaca of healthy birds, more than half were CR-negative (white colonies). No CR-negative (white) E. coli colonies were found in internal organs from birds with typical lesions of colisepticemia. We feel that these preliminary findings suggest that the CR dye binding could be used as a phenotypic marker to distinguish between invasive and noninvasive isolates.     

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.     

Rapid detection of virulence-associated genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction

Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.     

Characterization of extraintestinal Escherichia coli isolated from a peacock (Pavo cristatus) with colisepticemia

Extraintestinal infections by avian pathogenic strains of Escherichia coli (APEC) are commonly reported in poultry, but there is little information on infections by APEC in other bird species. Here we report on the characterization of extraintestinal E. coli isolated from a domesticated peacock, from the south of Brazil, that died of colisepticemia. Necropsy examination revealed congested liver, hypertrophied kidneys, peritonitis, severe typhlitis suggestive of coligranuloma, pneumonia, and airsacculitis--typical signs of colisepticemia. The isolates from lungs, kidney, heart, intestine, liver, and bone marrow all harbored the same virulence-associated factors (iucD, colV, iss, mat, fimC, ompA, traT crl, csgA vgrG, and hcp), yielded the same band pattern in amplified ribosomal DNA restriction analysis, and were allocated to the Escherichia coli Reference Collection group B1. The isolates were resistant to bacitracin, trimethoprim, and tetracycline, but displayed slight differences in their resistance to other antimicrobials. The isolates also differed in their virulence in 1-day-old chickens, but none displayed high virulence in vivo. We conclude that the peacock died of colisepticemia after it was infected with an extraintestinal E. coli strain of low virulence that nevertheless harbored virulence factors generally associated with APEC. This study represents the first characterization of an APEC isolated from a nonpoultry bird species.     

Persistence of cellulitis-associated Escherichia coli DNA fingerprints in successive broiler chicken flocks

Avian cellulitis in broiler chickens is primarily caused by Escherichia coli. Previous research found that the E. coli isolates of cellulitis origin were unique to each ranch, suggesting that these E. coli were endemic within the ranch environment. To test the hypothesis that the E. coli associated with cellulitis are endemic in the litter of the broiler house, we designed a study to determine whether E. coli DNA fingerprints associated with cellulitis persist over successive flocks that are grown in the same house. In addition, we assessed the impact of different cleaning and disinfection strategies on this persistence. Two broiler houses were followed on each of five farms over 3-4 flocks. A total of 353 E. coli isolates from cellulitis lesions were analyzed in this study, and 314 of these isolates (89%) were DNA fingerprinted by PFGE. In each ranch, there were several DNA fingerprint patterns that were present over successive flocks, regardless of the cleaning and disinfection strategy utilized. Isolates persisted as long as 191 days, implying that these E. coli are capable of persisting in the broiler house environment for long periods of time. In addition, these E. coli isolates were associated with cellulitis lesions in successive flocks. Thus, the isolates of E. coli that are associated with cellulitis in broiler chickens appear to be endemic in the litter environment of the broiler house.     

Shiga toxin genes in avian Escherichia coli

The purpose of this study was to determine the presence of stx genes in avian pathogenic Escherichia coli (APEC). We examined 97 APEC isolates: 34 from lesions of avian cellulitis, 31 from avian septicemia, 13 from swollen head syndrome (SHS) in chickens, and 19 from diseased turkeys. We also examined five isolates from the feces of healthy chickens. All 102 E. coli isolates were tested for the presence of stx genes by PCR amplification and by colony blots using probes specific for stx1 and stx2. Fifty-three percent (52) of the 97 APEC carried stx gene sequences: one isolate carried stx2 sequences, two carried both stx1 and stx2 sequences, and the remaining 49 isolates carried only stx1 sequences. Twenty-six isolates were positive by both hybridization and PCR amplification, 10 were positive by PCR only, and 16 were positive by hybridization only. All the stx-positive isolates were negative by PCR for the eae and E-hlyA genes. The five isolates from healthy chickens were all negative for stx. All 13 SHS isolates were positive for the stx1 gene and had low titres for cytotoxicity in the Vero cell assay (VCA). Other stx-positive isolates were negative in the VCA. The stx1 gene from one SHS E. coli isolate was cloned and sequenced and shown to be identical to that of the stx gene of Shigella dysenteriae. The observations indicate that stx1 gene sequences are widespread among APEC but that cytotoxicity on Vero cells is uncommon.     

Phenotypic and genotypic characterization of virulence factors of Escherichia coli isolated from broiler chickens with simultaneous occurrence of cellulitis and other colibacillosis lesions.

The objective of this study was to characterize virulence factors of Escherichia coli isolates from broilers with simultaneous occurrence of cellulitis and other colibacillosis lesions. Thirty flocks were sampled and 237 birds with cellulitis were examined. Eighty-two (34.6%) of 237 birds condemned for cellulitis had gross lesions in the heart, air sacs, joints, or liver. In 58 chickens, E. coli was isolated from both the cellulitis and other lesions of colibacillosis, and 18.9% of the E. coli isolates from the 2 types of lesions belonged to the same O group. Escherichia coli of serogroups O78, O1, and O2 predominated. Isolates of the same serogroup that were derived from different lesions in the same birds had similar patterns of biotype, aerobactin production, serum sensitivity profile, antibiotic sensitivity, and K1 capsule production. Escherichia coli derived from cellulitis lesions produced virulence factors similar to those found in E. coli isolated from other colibacillosis lesions in poultry.     

Genotypic and phenotypic characterization of potential virulence of intestinal avian Escherichia coli strains isolated in Algeria

In order to characterize potential pathogenic Escherichia coli strains isolated from diarrheic hens and chickens originating from intensive battery rearing in North Algeria, the presence of a large range of virulence factors and markers was studied in 50 strains by DNA-DNA hybridization on colonies and phenotypic tests. The sequences we focused on were those coding for adhesins F5, F41, F17, Pap, Afa, and Sfa; intimin Eae; and toxins STa, STb, LT1, Stx1, Stx2, CNF1, and CNF2. The phenotypes explored were the colicins, aerobactin, hemolysins, and hemagglutinin production and serum resistance. The genotypic and phenotypic tests enabled us to categorize the isolates into two distinct groups: those with a potential to invade the host (27 strains were serum resistant and/or produced aerobactin), among which three strains were also potentially diarrheagenic, one strain was LT1 + F17+ Afa+ Pap+ (enterotoxigenic E. coli) and the two others were Stx1 (verotoxigenic E. coli). Twenty-three strains were colicinogenic, including 19 strains producing colicin V. This latter factor was also detected in isolates negative for the other virulence factors. On the basis of the type of erythrocytes agglutinated, we established 14 mannose-resistant hemagglutination patterns among the 37 strains tested, including 22 serum-resistant and/or aerobactin producing strains and 15 strains negative for these two characters. None of the strains produced alpha hemolysin, whereas two strains produced beta hemolysin and enterohemolysin, respectively. Congo red fixation was observed in 25 strains. No relationship could be detected between Congo red fixation and the presence of other virulence markers, such as serum resistance and aerobactin production. This study shows that among isolates originating from the feces of diarrheic chickens, the proportion of potentially diarrheagenic E. coli strains is low.     

屠宰前鸡、猪源大肠杆菌耐药性调查

本试验旨在了解屠宰前鸡、猪源食品动物体内大肠杆菌耐药情况,分析潜在的食品安全问题。从广州市畜禽交易市场随机采集待屠宰鸡和猪的粪便样品,分离鉴定大肠杆菌,并采用琼脂稀释法检测大肠杆菌对15种抗菌药物的敏感性。结果显示,从658份猪源样品和133份鸡源样品中共分离鉴定出731株大肠杆菌,其中猪源606株,鸡源125株。药敏试验结果显示,731株大肠杆菌均表现出不同程度的耐药,耐药谱广且多重耐药现象严重。对复方新诺明和四环素的耐药率为90.0%以上,仅对头孢西丁、黏菌素和阿米卡星较敏感(耐药率均低于3%)。鸡源大肠杆菌对头孢噻肟、头孢曲松、新霉素、阿米卡星、萘啶酸和环丙沙星的耐药率显著高于猪源大肠杆菌(P＜0.05)。鸡源大肠杆菌中3耐及3耐以上的菌株占97.60%,猪源大肠杆菌占94.72%。结果表明,屠宰前畜禽体内大肠杆菌对临床常用抗菌药物的耐药性非常严重,以多重耐药为主,且耐药谱丰富多样。提示屠宰前畜禽携带的耐药菌对食品安全和人类健康存在较大的安全隐患。     

Pathotypes of avian Escherichia coli as related to tsh-, pap-, pil-, and iuc-DNA sequences, and antibiotic sensitivity of isolates from internal tissues and the cloacae of broilers

One hundred four Escherichia coli isolates were collected from internal tissues and the cloacae of broilers with colibacillosis or from the cloacae of healthy birds. The isolates were tested for the presence of DNA sequences for temperature-sensitive hemagglutinin (tsh), for P (pap) and F1 (pil) fimbriae, and for aerobactin synthesis (iuc) by DNA/DNA hybridization. The isolates were also tested for O1, O2, and O78 serogroups, serum and antibiotic resistance, and virulence in day-old chickens. The Tsh/Pil/Iuc was the major pathotype detected in 53.8% of isolates from internal tissues, as compared with only 28.8% of isolates from the cloacae. The Tsh/Pap/Iuc pathotype was detected at a lower frequency (15.4%) but only in isolates from internal tissues. Among the virulence-associated marker genes, tsh and iuc were detected in most of the isolates from internal tissues (90.4% and 92.3%), as compared with only 51.9% and 63.5% of isolates from the cloacae, respectively, pap was detected to a lesser extent, in 25% of isolates but only from internal tissues. In contrast to the pil gene, the tsh-, pap-, and iuc-DNA sequences were more frequently detected in isolates from internal tissues than in isolates from the cloacae. O-antigen typing revealed that 25% of isolates belonged to serogroups O1 (4.8%), O2 (9.6%), and O78 (10.6%). Although most isolates appeared to be resistant to serum, only isolates from internal tissues were virulent in day-old chickens in contrast to isolates from the cloacae. More than 10% of isolates were resistant to most of the antibiotics used for the study. However, less resistance to enrofloxacin and norfloxacin was observed. Our data suggest that the Tsh/Pil/Iuc and Tsh/Pap/Iuc pathotypes and Tsh and Iuc virulence-associated markers are important factors of avian pathogenic E. coli. Enrofloxacin appeared to be the best choice for treatment of the infection.