Characterization of Escherichia coli isolated from healthy dogs

Five month old dogs from a Midwestern research kennel occasionally developed bloody diarrhea after shipment to other facilities. As previous diagnostic efforts failed to reveal any potential pathogens in feces from normal and diarrheic dogs, Escherichia coli was investigated for select virulence properties that may contribute to the occurrence of bloody diarrhea. Fecal swabs from 52 healthy dogs were examined for E. coli. Two hundred and sixty E. coli-like colonies were screened by PCR for the attaching and effacing (eae) gene, Shiga toxin (stx) genes, and the heat-stable enterotoxin type A (sta) gene. One hundred forty two of the 260 E. coli-like colonies (54.6%) from 43 dogs were eae or sta positive; and 60 of the eae and/or sta positive isolates were examined further. Among the 60 isolates, 23 (38.3%) possessed the eae gene, 32 (53.3%) possessed the sta gene, and five (8.3%) possessed both eae and sta genes (eae⁺/sta⁺). Of the 60 isolates, six sta⁺ and one eae⁺/sta⁺ isolates were hemolytic. When examined in the suckling mouse assay, five of six sta⁺ isolates and three of four eae⁺/sta⁺ isolates gave gut-to-remaining carcass ratios ≥0.083, indicating expression of heat-stable enterotoxin. These enterotoxin-producing isolates belonged to serogroups O42, O170, and O-negative.     

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.     

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.     

Dynamics of shiga-toxin producing Escherichia coli (STEC) and their virulence factors in cattle

Starting at birth, twenty Holstein calves were housed individually, in groups of five and finally in one large freestall while fecal samples were collected weekly for 25 weeks. From each sample, twenty isolates of Escherichia coli were screened for 6 virulence markers including shiga-toxin 1, 2, intimin, enterohemolysin, the fimbrial antigen efa1 and the adhesin saa. Dynamic models of transmission of E. coli were used to model the transmission of different virulotypes between calves and the loss of the same virulotypes from the calves. It was found that, once E. coli encoding shiga-toxins in combination with enterohemolysin were transmitted and established in a calf, they tended to be eliminated less efficiently compared to E. coli without this combination of virulence markers. It was concluded that the presence of certain combinations of virulence markers coincided with persistence of E. coli in the bovine gastrointestinal tract. In addition, the combinations of stx with either eae or ehxA in E. coli have a greater impact on the loss rates than on the transmission rates.     

Genotypic identification of Pseudomonas aeruginosa strains isolated from patients with urinary tract infection

BackgroundNumerous nosocomial infections including urinary tract infection (UTI) have been reported to be linked to Pseudomonas aeruginosa (P. aeruginosa). This bacterium is one of the most common pathogen colonized in the urinary tract. The main purpose of this study was to evaluated the presence of antibiotic resistance genes and also the most frequent genotype patterns of P. aeruginosa in the patients with UTI hospitalized in different wards of hospitals.Materials and methodsIn this study, 70 strains of P. aeruginosa isolated of urine samples from the patients with UTI were assessed. The isolated strains were genotyped using Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) method. We have also analyzed the presence of TEM and SHV resistant genes in the isolates.ResultsA total of 70 P. aeruginosa strains was isolated from the UTI patients. Based on MLVA method, 61 various genotypes of P. aeruginosa were identified which grouped into two main clusters and 4 sub-clusters. Moreover, approximately 80% and 70% of isolated strains carried the TEM and SHV resistance genes, respectively.ConclusionOur findings showed that the majority of patients hospitalized in different wards of hospitals have experienced the urinary tract infection caused by P. aeruginosa. According to the genotyping results, a high diversity of the P. aeruginosa population was observed in the patients with UTI. Our results can provide a better understanding of the P. aeruginosa genotype distribution and epidemiology of infection, which can be applied as basic data for future antibiotic therapies.     

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.     

traT and CNF2 genes of Escherichia coli isolated from milk of healthy cows and sheep

The objectives of the present study were to isolate Escherichia coli from milk of apparently healthy cows and sheep and to investigate the presence of traT and cytotoxic necrotising factor-2 (CNF2) virulence genes by multiplex polymerase chain reaction (PCR). Milk samples collected from a total of 1028 apparently healthy ruminants (737 cows and 291 sheep) in eastern Turkey were streaked onto 5% sheep-blood agar. E. coli was isolated and identified by biochemical tests in 5.9% (61/1028) of milk samples. Correct amplification with the molecular length of 232 bp was obtained from all E. coli isolates by the species-specific PCR. The isolation rates of the agent were calculated to be 7.6% (56/737) in cows and 1.7% (5/291) in sheep. The difference between these proportions was statistically significant (p < 0.001). Multiplex PCR showed that traT and CNF2 genes were present in 62.3% and 6.6% of all isolates, respectively. Both genes were present in 16.4% of the isolates, with only 14.7% having neither gene.     

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.     

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.     

Characterisation of Escherichia fergusonii isolates from farm animals using an Escherichia coli virulence gene array and tissue culture adherence assays

Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of E. fergusonii collected between 2003 and 2004 were screened using an Escherichia coli virulence gene array to test for the presence of homologous virulence genes in E. fergusonii. The iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the prfB (P-related fimbriae regulatory) and ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the E. fergusoniiprfB, iss and ireA genes shared a high degree of sequence similarity to their counterparts in E. coli, and a particular resemblance was noted with the E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine E. fergusonii isolates associated with HEp-2 cells with a ‘localised adherence’ or ‘diffuse adherence’ phenotype, and they proved to be moderately invasive. The E. fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of E. coli, and support existing evidence that strains of E. fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored.     

Virulence factors and antimicrobial susceptibility of Escherichia coli isolated from calves in Turkey

Escherichia coli isolates from calves were investigated by multiplex PCR assays for the presence of genes encoding K99, F41, F17-related fimbriae, heat-stabile enterotoxin a (STa), intimin (eae) and Shiga toxins (stx1 and stx2). A total of 120 E. coli isolates, 75 isolated from diarrhoeic or septicemic calves and 45 from clinically healthy calves aged between 1 day and 2 months were tested. Each isolate was obtained from different calves in different herds. Among the isolates from diseased animals, 12 (16%) isolates from 1- to 7-day-old diarrhoeic calves were detected as enterotoxigenic E. coli which possessed K99, F41 and STa in combination; F17-related fimbriae genes were detected in 33 (44%) isolates and they were found in combination with K99 + F41 + STa in two isolates. Of 120 isolates, 16 carried eae, eight stx1 and five stx2 genes alone or in combination. None of the eae- or stx-positive strains was identified as O157:H7. However, results indicate that calves may be carrier of Shiga toxin-producing E. coli which have potential as a human pathogen. Antimicrobial susceptibility of 75 isolates from diseased calves was determined by agar disk diffusion method for 14 antimicrobial agents. In 77.3% of the isolates, multiresistance was detected. Higher resistance rates were detected for cephalothin (72%), tetracycline (69.3%), kanamycin (69.3%), ampicillin (65.3%), nalidixic acid (53.3%), trimethoprim-sulphamethoxazole (52%) and enrofloxacin (41.3%), respectively. No resistance was found for ceftiofur and cefoxitin.     

Phylogenetic distribution of virulence genes in Escherichia coli isolated from bovine mastitis in Iran

One hundred and twenty seven Escherichia coli isolates from bovine mastitis were examined to detect the phylogenetic group/subgroups and a selection of virulence associated genes. Forty nine (38.58%) isolates belonged to group B1 the remaining isolates fell into four phylogenetic subgroups: A₀ (18.11%), A₁ (26.77%), D₁ (6.29%) and D₂ (10.23%). None of the isolates belonged to B2 group. Forty seven (37.00%) isolates were positive for at least one virulence gene, among them f17A was the most common gene, found in 20.47% of the isolates. Among the E. coli isolates, 11.81% had iucD, 9.44% f17c-A, 9.44% cnf2, 7.87% f17b-A, 6.29% afaD-8 and afaE-8, 3.14% f17d-A, 0.78% cnf1 and 0.78% clpG genes. All of the detected virulence genes were present alone or in combination with each other except clpG and f17d-A genes that were only found alone. None of the isolates contained the genes for F17a-A, intimin, P or S fimbriae.     

Absence of urinary tract infection in dogs with experimentally induced hyperadrenocorticism

Objectives of this study were to determine occurrence of urinary tract infection and describe results of urine analysis and urine culture in dogs with experimentally induced hyperadrenocorticism. Dogs were randomly assigned to receive either hydrocortisone (nine dogs) or placebo (eight dogs) for 49 consecutive days. Before and on day 49 of treatment, evaluation of dogs included physical examination, abdominal ultrasound, urine culture, urinalysis, adrenal function testing, and measurement of urine protein and creatinine and activity of serum alkaline phosphatase. All dogs in the experimental group had clinical and laboratory findings of hyperadrenocorticism. Urine specific gravity was significantly decreased and urine protein-to-creatinine ratio was significantly increased in dogs with hyperadrenocorticism. Urinary tract infection did not occur in any dogs. We conclude that administration of hydrocortisone created a model of hyperadrenocorticism; however, urinary tract infection did not occur. Additional evaluation is needed to determine association between urinary tract infection and hyperadrenocorticism.     

牦牛产志贺毒素大肠杆菌主要黏附因子相关基因的分子流行病学调查

为了解中国牦牛产志贺毒素的大肠杆菌(Shiga toxin-producing Escherichia coli, STEC)中主要黏附因子的流行情况,采用PCR方法对来自四川甘孜阿坝等地区健康牦牛的70株STEC的eae、saa、iha 3种与黏附相关的毒力基因进行检测,并对部分含有相关黏附因子的阳性分离株的毒力基因进行了克隆及序列分析。结果显示,牦牛STEC中saa、iha的阳性率分别为71.42%(50/70)和78.57%(55/70),无eae基因序列(0/70),saa、iha的测序结果与GenBank上序列的同源性分别为100%和93%~99%。健康牦牛分离的STEC无LEE毒力岛编码eae,其他的一些与黏附相关的主要毒力基因saa、iha的携带率较高。     

Uropathogenic virulence factors in isolates of Escherichia coli from clinical cases of canine pyometra and feces of healthy bitches

Escherichia coli is commonly isolated in canine pyometra, but little is known of the virulence factors that may be involved in the precipitation of this disease. The aim of this study was to compare the prevalence of uropathogenic virulence factor (UVF) genes in E. coli isolates from canine pyometra and from feces of healthy bitches to evaluate their role in the pathogenesis of pyometra. E. coli from 23 cases of canine pyometra and from the feces of 24 healthy bitches were analyzed, by polymerase chain reaction, for UVF genes associated with canine and human urinary tract infections (UTIs). The prevalences of UVFs in E. coli from canine pyometra were similar to that in canine and human uropathogenic E. coli. The prevalence of pap was greater (P=0.036) for E. coli from pyometra (52%) than for fecal isolates (21%), and the papGIII allele was present in all pap-containing isolates. The prevalences of genes for alpha-haemolysin and cytotoxic necrotising factor 1 were not significantly higher (P=0.075) in E. coli from pyometra than from feces. The proportion of pyometra strains with >or=3 UVFs was higher (P=0.039) than that of fecal strains, suggesting that possession of >or=3 UVF genes enhances the pathogenicity of the strain. Our findings demonstrate that E. coli associated with canine pyometra are similar to uropathogenic strains, and that operons that encode P fimbriae, alpha-haemolysin and cytotoxic necrotising factor 1 probably enhance the virulence and pathogenicity of the strain in the canine genital tract.     

Bacterial species isolated from cats with lower urinary tract infection and their susceptibilities to cefovecin

Background

The aim of this study was to determine the bacterial species recovered from 61 cats with lower urinary tract infection (LUTI), and their susceptibility to cefovecin in vitro.

Results

The clinical signs and final clinical diagnosis for cats with confirmed LUTI were also reported. After physical examination of the cats, urine samples including ≥5-6 leucocytes in microscopic evaluation were cultured using bacteriological techniques. The isolates were identified by conventional microbiological methods and tested for in vitro susceptibility using the Kirby-Bauer disc diffusion method recommended by the Clinical Laboratory Standards Institute. Bacterial growth was observed in 16 of 61 urine samples. Antimicrobial susceptibility tests showed that 13 of 16 (81%) isolates were susceptible to cefovecin. The most frequently isolated bacterium from cats with signs of lower urinary tract infection, was Escherichia coli.

Conclusion

Cefovecin was found to be effective in cats with LUTI. Because cefovecin is a new antimicrobial agent in veterinary medicine, there are only few studies about urine culture of cats with LUTI. It is the first study on in vitro activity of cefovecin against bacterial isolates from cats with lower urinary infections in Istanbul, Turkey.     

石河子地区隐性乳房炎奶牛源大肠杆菌的分离鉴定及耐药性分析

为了解引起石河子地区规模化奶牛场隐性乳房炎奶牛源大肠杆菌的流行情况及其耐药性,本试验采用兰州乳房炎检测板(Lanzhou mastitis test,LMT)对石河子地区11个规模化奶牛场进行了检测,同时对无菌采集的检测为阳性的乳样进行了大肠杆菌分离鉴定及耐药性分析.结果显示,石河子地区11个规模化奶牛场的隐性乳房炎感染阳性率为44.56%(313/1950);对313份无菌采集的乳样进行细菌分离鉴定,结果共分离出病原菌108株,其中大肠杆分离率为14.81%(16/108);药敏试验结果显示16株大肠杆菌对氧氟沙星、头孢唑啉、克林霉素高度敏感,对庆大霉素、氨苄西林、链霉素中度敏感,对阿莫西林、四环素有耐药性.本试验为该地区大肠杆菌源性隐性乳房炎的防制提供科学依据.     

Detection of genes encoding for virulence and adherence factors in Escherichia coli isolated in slaughtered Sarda breed sheep

In order to investigate the pathogenic profile of Escherichia coli hosted in “Sarda” sheep, autochthonous race present in Sardinia, thirty-seven E. coli strains collected from different sources (fleeces, carcass swabs and gut mucosa) of pre-chill slaughtered sheep (ewes and lambs) were serotyped using pheno- and genotypic methods. Furthermore, the presence of genes encoding for virulence factors and mediating for localized mucosal adherence factors was investigated, and pulsed-field gel electrophoresis (PFGE) characterization was performed.     

Phylogenetic grouping,epidemiological typing,analysis of virulence genes,and antimicrobial susceptibility of Escherichia coli isolated from healthy broilers in Japan

Background

The aim of our study was to investigate the possible etiology of avian colibacillosis by examining Escherichia coli isolates from fecal samples of healthy broilers.

Findings

Seventy-eight E. coli isolates from fecal samples of healthy broilers in Japan were subjected to analysis of phylogenetic background, virulence-associated gene profiling, multi-locus sequence typing (MLST), and antimicrobial resistance profiling. Phylogenetic analysis demonstrated that 35 of the 78 isolates belonged to group A, 28 to group B1, one to group B2, and 14 to group D. Virulence-associated genes iutA, iss, cvaC, tsh, iroN, ompT, and hlyF were found in 23 isolates (29.5%), 16 isolates (20.5%), nine isolates (11.5%), five isolates (6.4%), 19 isolates (24.4%), 23 isolates (29.5%), and 22 isolates (28.2%) respectively. Although the genetic diversity of group D isolates was revealed by MLST, the group D isolates harbored iutA (10 isolates, 71.4%), iss (6 isolates, 42.9%), cvaC (5 isolates, 35.7%), tsh (3 isolates, 21.4%), hlyF (9 isolates, 64.3%), iroN (7 isolates, 50.0%), and ompT (9 isolates, 64.3%).

Conclusions

Our results indicated that E. coli isolates inhabiting the intestines of healthy broilers pose a potential risk of causing avian colibacillosis.