Pathotyping avian pathogenic Escherichia coli strains in Korea

To examine the genetic background of avian pathogenic Escherichia coli (APEC) that affects virulence of this microorganism, we characterized the virulence genes of 101 APEC strains isolated from infected chickens between 1985~2005. Serotypes were determined with available anti-sera and median lethal doses were determined in subcutaneously inoculated chicks. The virulence genes we tested included ones encoding type 1 fimbriae (fimC), iron uptake-related (iroN, irp2, iucD, and fyuA), toxins (lt, st, stx1, stx2, and vat), and other factors (tsh, hlyF, ompT, and iss). Twenty-eight strains were found to be O1 (2.0%), O18 (3.0%), O20 (1.0%), O78 (19.8%), and O115 (2.0%) serotypes. The iroN (100%) gene was observed most frequently followed by ompT (94.1%), fimC (90.1%), hlyF (87.1%), iss (78.2%), iucD (73.3%), tsh (61.4%), fyuA (44.6%), and irp2 (43.6%). The strains were negative for all toxin genes except for vat (10.9%). All the strains were classified into 27 molecular pathotypes (MPs). The MP25, MP19, and MP10 pathotypes possessing iroN-fimC-ompT-hlyF-iucD-tsh-iss-irp2-fyuA (22.8%), iroN-fimC-ompT-hlyF-iucD-tsh-iss (21.8%), and iroN-fimC-ompT-hlyF-iss (11.9%) genotypes, respectively, were predominant. Redundancy of iron uptake-related genes was clearly observed and some strains were associated with higher mortality than others. Therefore, strains with the predominant genotypes can be used for diagnosis and vaccine.     

The expression of plasmid mediated afimbrial adhesin genes in an avian septicemic Escherichia coli strain

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD₅₀ of 4.0 × 10⁵ CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD₅₀ of 1.2 × 10¹² CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes.     

致病性鸡大肠杆菌type1菌毛fimC基因的克隆与序列测定

根据国外发表的人致病性大肠杆菌fimC基因序列,在其保守区设计了带有BamHⅠ/HindⅢ酶切位点的一对引物,应用PCR技术以鸡致病性大肠杆菌O2基因组DNA为模板扩增到一个片段,大小约为700bp,将扩增产物克隆到pMD18-T载体上,并转化于大肠杆菌TG1宿主菌,经酶切和筛选,得到阳性重组质粒.通过对阳性重组质粒核酸序列测定,确定此DNA片段为鸡大肠杆菌fimC基因.     

Molecular characterization of Escherichia coli O157:H7 strains isolated from different sources and geographic regions

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.     

Virulence factors and genetic variability of uropathogenic Escherichia coli isolated from dogs and cats in Italy

In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between α-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.     

Extraintestinal pathogenic Escherichia coli in poultry meat products on the Finnish retail market

Background

Extraintestinal pathogenic Escherichia coli bacteria (ExPEC) exist as commensals in the human intestines and can infect extraintestinal sites and cause septicemia. The transfer of ExPEC from poultry to humans and the role of poultry meat as a source of ExPEC in human disease have been discussed previously. The aim of the present study was to provide insight into the properties of ExPEC in poultry meat products on the Finnish retail market with special attention to their prevalence, virulence and phylogenetic profiles. Furthermore, the isolates were screened for possible ESBL producers and their resistance to nalidixic acid and ciprofloxacin was tested.

Methods

The presence of ExPEC in 219 marinated and non-marinated raw poultry meat products from retail shops has been analyzed. One E. coli strain per product was analyzed further for phylogenetic groups and possession of ten virulence genes associated with ExPEC bacteria (kpsMT K1, ibeA, astA, iss, irp2, papC, iucD, tsh, vat and cva/cv) using PCR methods. The E. coli strains were also screened phenotypically for the production of extended-spectrum β-lactamase (ESBL) and the susceptibility of 48 potential ExPEC isolates for nalidixic acid and ciprofloxacin was tested.

Results

E. coli was isolated from 207 (94.5%) of 219 poultry meat products. The most common phylogenetic groups were D (50.7%), A (37.7%), and B2 (7.7%). Based on virulence factor gene PCR, 23.2% of the strains were classified as ExPEC. Two ExPEC strains (1%) belonged to [O1] B2 svg+ (specific for virulent subgroup) group, which has been implicated in multiple forms of ExPEC disease. None of the ExPEC strains was resistant to ciprofloxacin or cephalosporins. One isolate (2.1%) showed resistance to nalidixic acid.

Conclusions

Potential ExPEC bacteria were found in 22% of marinated and non-marinated poultry meat products on the Finnish retail market and 0.9% were contaminated with E. coli [O1] B2 svg+ group. Marinades did not have an effect on the survival of ExPEC as strains from marinated and non-marinated meat products were equally often classified as ExPEC. Poultry meat products on the Finnish retail market may have zoonotic potential.     

Occurrence of weak mutators among avian pathogenic Escherichia coli (APEC) isolates causing salpingitis and peritonitis in broiler breeders

A collection of 46 avian pathogenic Escherichia coli (APEC) isolates was examined for the presence of mutators by determining the rate of mutation to rifampicin resistance. The collection included 34 E. coli isolates obtained in pure culture from chronic lesions of salpingitis and peritonitis in 34 broiler breeders, of which 12 were associated with the development of secondary septicemia. Twelve additional isolates were obtained from a clonal outbreak (ST95) of E. coli peritonitis syndrome (EPS), the lesions of which changed gradually over time into a subacute/chronic form. The hypothesis of the present study was that mutation rates would be higher for chronic infection isolates than for isolates from acute infections/exacerbations. The distribution of mutation rates followed a pattern similar to that found for other clinical isolates of E. coli, with a modal/median value of 1.47 × 10⁻⁸. Of the 46 isolates, 24% (n = 11) were weakly hypermutable (2.00 × 10⁻⁸ ≤ μ < 2.00 × 10⁻⁷), however, no strong mutators were detected (μ ≥ 2.00 × 10⁻⁷). Chronic salpingitis isolates had the highest proportion (45%, P = 0.001) of weak mutators and also, significantly higher mutation rates (P = 0.003) compared to isolates that caused septicemia (4%). In addition, mutation rates were significantly lower among ST95 isolates (P < 0.0005), and among isolates from the same clonal group as ST95 (P = 0.027), when compared to isolates from other groups. Although a clear association with the time phase of infection (as lesions of EPS became more chronic) could not be observed (ρ = 0.523, P = 0.081), a higher frequency of weak mutators among chronic infection isolates suggests that increased mutation rates play a role in adaptation of APEC to long-term persistence in an infected host environment.     

应用DNA芯片技术筛选禽致病性大肠杆菌和尿道致病性大肠杆菌的差异表达基因

为研究禽致病性大肠杆菌强毒株E058的毒力相关基因在鸡体内、体外表达情况以及E058和尿道致病性大肠杆菌HEC4在LB和尿液中培养的表达情况,本研究分别提取E058株在SPF鸡体内及E058株和HEC4株在LB和尿液中静置培养的总RNA,与构建的DNA芯片杂交,检测和分析RNA的差异表达情况。芯片的检测结果表明:E058株在鸡体内和LB中培养差异表达基因共有9个,上调基因为5个,分别为neuC、iutA、cvaC、aes-15和iucCD;下调基因为4个,分别为aes-8、gyrB、aec-30和mdh。另外,芯片检测结果也显示E058株和HEC4株在LB和尿液中静置培养,具有相似的基因表达情况。     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) from cattle in Korea between 2010 and 2011

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.     

Characterisation of avian pathogenic Escherichia coli (APEC) associated with colisepticaemia compared to faecal isolates from healthy birds

A total of 114 avian pathogenic Escherichia coli (APEC) isolates were collected from cases of colisepticaemia occurring in broilers (77) and layers (37) within Ireland. In addition 45 strains isolated from faeces of healthy birds were included for comparison. All isolates were serogrouped, and examined for known virulence factors, mostly by PCR. The O78 serogroup represented 55 and 27% of broiler and layer colisepticaemic isolates respectively. All isolates were positive for curli fimbriae (crl, csg) and negative for afimbrial adhesin (afa). S-fimbrial (sfa) sequences were present in 8.8% of septicaemic isolates and 8.9% of healthy bird isolates. The majority of E. coli from cases of colisepticaemia (97.4%) and healthy bird (95.6%) isolates were positive for aerobactin (aer), and temperature sensitive haemagglutinin (tsh) was similarly detected in high numbers in 93.9 and 93.3%, respectively. In comparison to E. coli isolates from the faeces of healthy birds, a significantly higher percentage of isolates from septicaemic cases possessed Type 1 fimbriae (fimC) and increased serum survival (iss) gene sequences. Forty-seven (41.2%) isolates from septicaemic birds possessed P-fimbriae (pap) gene sequences, compared with only 15.6% from E. coli isolated from healthy birds. Haemolysin (hlyE) sequences were detected in 46.7% of isolates from healthy birds in comparison with 6.1% of septicaemic isolates. Sequences encoding colicin V (cvaC) were detected in 99.1% of septicaemic isolates and 82.2% of isolates from healthy birds. The K1 capsule was only present in two septicaemic isolates, both taken from layers. Motility was detected in 36.8% of E. coli isolated from cases of septicaemia, compared with 93.3% of isolates from healthy birds. These results demonstrate the presence of 11 virulence genes in E. coli isolated from cases of colisepticaemia within Ireland, and indicate the prevalence of iss and fimC.     

Virulence factors in Escherichia coli isolated from calves with diarrhea in Vietnam

This study was conducted to determine the prevalence and characteristics of pathogenic Escherichia (E.) coli strains from diarrheic calves in Vietnam. A total of 345 E. coli isolates obtained from 322 diarrheic calves were subjected to PCR and multiplex PCR for detection of the f5, f41, f17, eae, sta, lt, stx1, and stx2 genes. Of the 345 isolates, 108 (31.3%) carried at least one fimbrial gene. Of these 108 isolates, 50 carried genes for Shiga toxin and one possessed genes for both enterotoxin and Shiga toxin. The eae gene was found in 34 isolates (9.8%), 23 of which also carried stx genes. The Shiga toxin genes were detected in 177 isolates (51.3%) and the number of strains that carried stx1, stx2 and stx1/stx2 were 46, 73 and 58, respectively. Among 177 Shiga toxin-producing E. coli isolates, 89 carried the ehxA gene and 87 possessed the saa gene. Further characterization of the stx subtypes showed that among 104 stx1-positive isolates, 58 were the stx1c variant and 46 were the stx1 variant. Of the 131 stx2-positive strains, 48 were stx2, 48 were stx2c, 11 were stx2d, 17 were stx2g, and seven were stx2c/stx2g subtypes. The serogroups most prevalent among the 345 isolates were O15, O20, O103 and O157.     

禽致病性大肠杆菌ibeA基因缺失及其特性分析

运用大肠杆菌Red同源重组系统,对禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)DE02株的ibeA基因进行了缺失,其结果为揭示IbeA的功能奠定了基础.通过对APEC ibeA基因缺失株与人脑微血管内皮细胞(Human brain microvascular endothelial cells,HBMECs)的黏附与侵袭,发现ibeA基因缺失后与HBMECs的黏附率为45%,侵袭率为1.2%,相对于野生型APEC均显著降低;提示ibeA基因是APEC的重要致病因子.     

Virulence factors in Escherichia coli strains isolated from urinary tract infection and pyometra cases and from feces of healthy dogs

The aim of this study was to compare the prevalence of virulence genes in 158 Escherichia coli strains isolated from 51 clinical cases of UTIs, 52 of pyometra and from 55 fecal samples from healthy dogs by PCR. papC was found in 12 (23.5%) strains isolated from UTIs, 19 (36.5%) from pyometra and 10 (18.2%) from feces. papGII was observed in 3 (5.8%) strains from pyometra, and papGIII in 10 (19.6%) from UTIs, 15 (28.8%) from pyometra and 9 (16.4%) from feces. sfaS was detected in 22 (43.1%) strains from UTIs, 24 (46.1%) from pyometra and 19 (34.5%) from feces. hlyA was observed in 17 (33.3%) strains from UTIs, 18 (34.6%) from pyometra and 7 (12.7%) from feces, while cnf-1 was detected in 11 (21.6%) from UTIs, 21 (40.4%) from pyometra and 9 (16.4%) from feces. iucD was observed in 12 (23.5%) strains from UTIs, 9 (17.3%) from pyometra and 1 (1.8%) from feces. usp was found 17 (33.3%) isolates from UTIs and 36 (69.9%) from pyometra.     

Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplified polymorphic DNA (RAPD) analysis

Here we describe the application of a random amplified polymorphic DNA (RAPD) analysis for molecular genetic typing avian pathogenic Escherichia coli (APEC) strains. The RAPD technique was shown to be highly reproducible. Stable banding patterns with a high discriminatory capacity were obtained using two different primers. Overall, 55 E. coli strains were analyzed with a RAPD technique. The RAPD analysis showed that the E. coli strains isolated from poultry in Thailand and Sweden could be grouped into 50 of RAPD types by using these two different primer sets. Most of these different E. coli RAPD types were not geographically restricted. There was, as expected, a tendency of higher genetic relationship among E. coli strains isolated from the same farm. It is suggested that the RAPD technique may provide a rapid, low cost, simple and powerful tool to study the clonal epidemiology of avian E. coli infections.     

Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry

The molecular biology and epidemiology of 150 avian pathogenic Escherichia coli strains (APEC) isolated from septicemic poultry in Germany was investigated by serotyping, pulsed field gel electrophoresis (PFGE), and polymerase chain reaction (PCR). Only 49.6% of the isolates could be grouped to serogroups O1, O2, and O78. Macrorestriction analyses data revealed two large clonal groups (clusters I and II) among the APEC strains with a similarity of 60.9% to each other. An association between restriction pattern and serogroup or origin of the strains was only present in a few subgroups of each clusters I and II, but was not evident. In contrast, our data revealed distinct combinations of virulence-associated genes in that 51.2% of the O2-strains harboured a combination of the genes fyuA, irp2, iucD, tsh, vat, fimC, and colV and 36.4% of the O78-strains possessed the same gene combination with exception of vat. With 34 different gene combinations the non-O1, -O2, -O78 isolates revealed a higher variability in their virulence gene pattern than O1-, O2-, and O78-strains with 6, 13, and 9 patterns, respectively. Our data indicate only a limited association between the virulence gene pattern and the serogroup of APEC strains and question the sensitivity of O-typing for APEC identification without the application of further diagnostic tools. Although a limited number of APEC clones exist, horizontal gene transfer seems to be common in these pathogens. These findings strengthen further research on the population structure of APEC and may be the reason for the lack of clear definition of this common E. coli pathotype.     

Antimicrobial resistance of Escherichia coli isolates from canine urinary tract infections

This study determined the antimicrobial resistance profiles of Escherichia coli isolates from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 201 dogs with UTI diagnosed through clinical examination and urinalysis were processed for isolation of Escherichia coli. Colonies from pure cultures were identified by biochemical reactions (n=114) and were tested for susceptibility to 18 antimicrobials. The two most frequent antimicrobials showing resistance in Urinary E. coli isolates were oxytetracycline and ampicillin. Among the resistant isolates, 17 resistance patterns were observed, with 12 patterns involving multidrug resistance (MDR). Of the 69 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 50.9% of the isolates, whereas the remaining 25.5% isolates carried the tet(A) determinant. Most ampicillin and/or amoxicillin-resistant E. coli isolates carried bla_TEM-1 genes. Class 1 integrons were prevalent (28.9%) and contained previously described gene cassettes that are implicated primarily in resistance to aminoglycosides and trimethoprim (dfrA1, dfrA17-aadA5). Of the 44 quinolone-resistant E. coli isolates, 38 were resistant to nalidixic acid, and 6 were resistant to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in the GyrA (Ser83Leu) and ParC (Ser80Ile) genes. Furthermore, the aminoglycoside resistance gene aacC2, the chloramphenicol resistant gene cmlA and the florfenicol resistant gene floR were also identified. This study revealed an alarming rate of antimicrobial resistance among E. coli isolates from dogs with UTIs.     

Isolation and characterization of antimicrobial-resistant Escherichia coli from national horse racetracks and private horse-riding courses in Korea

Limited information is available regarding horse-associated antimicrobial resistant (AR) Escherichia (E.) coli. This study was designed to evaluate the frequency and characterize the pattern of AR E. coli from healthy horse-associated samples. A total of 143 E. coli (4.6%) were isolated from 3,078 samples collected from three national racetracks and 14 private horse-riding courses in Korea. Thirty of the E. coli isolates (21%) showed antimicrobial resistance to at least one antimicrobial agent, and four of the AR E. coli (13.3%) were defined as multi-drug resistance. Most of the AR E. coli harbored AR genes corresponding to their antimicrobial resistance phenotypes. Four of the AR E. coli carried class 1 integrase gene (intI1), a gene associated with multi-drug resistance. Pulsed-field gel electrophoretic analysis showed no genetic relatedness among AR E. coli isolated from different facilities; however, cross-transmissions between horses or horses and environments were detected in two facilities. Although cross-transmission of AR E. coli in horses and their environments was generally low, our study suggests a risk of transmission of AR bacteria between horses and humans. Further studies are needed to evaluate the risk of possible transmission of horse-associated AR bacteria to human communities through horse riders and horse-care workers.     

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.     

四黄止痢复方醇提物(CSME)抗禽大肠杆菌的作用机制

为了探讨四黄止痢复方醇提物(CSME)对禽大肠杆菌的抗菌机制。采用微孔-平板法检测其对禽大肠杆菌的最小抑菌浓度(MIC);用吸光光度法检测其对细菌生长曲线、细胞膜通透性和能量代谢活力的影响;荧光定量PCR法检测其对禽大肠杆菌4种主要毒力因子(tsh、iss、ibeA和fimC)表达的影响。结果显示,CSME对禽大肠杆菌(CVCC251)的MIC为125 g/L,1/2MIC就能显著抑制细菌生长,破坏细菌细胞膜结构,使核酸、蛋白质外泄,并抑制了琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)的活性;1/8MIC能显著抑制了禽大肠杆菌tsh、iss和fimC毒力因子基因的表达(P<0.01)。结果表明,CSME可以通过破坏细菌细胞膜结构、造成细菌能量代谢紊乱、抑制毒力基因表达发挥抗菌活性。