Detection of Shiga Toxin‐Producing Escherichia coli and Enteropathogenic Escherichia coli in Faecal Samples of Healthy Mithun (Bos frontalis) by Multiplex Polymerase Chain Reaction

Faecal samples obtained from 190 healthy mithuns were examined for the presence of Escherichia coli. Total one‐hundred and five E. coli isolates were obtained from these samples, which belonged to 55 different serogroups. These isolates were subjected to multiplex polymerase chain reaction (m‐PCR) for detection of stx1, stx2, eaeA and hlyA genes. Twenty‐three (21.90%) E. coli isolates belonging to 14 serogroups revealed the presence of at least one virulence gene when examined by m‐PCR. Nineteen percent and 2.85% of the mithuns were found to carry Shiga toxin‐producing E. coli (STEC) and enteropathogenic E. coli, respectively. stx1 and stx2 genes were found to be prevalent in 7 (6.67%) and 18 (17.14%) of the isolates respectively, whereas eaeA and hlyA genes were found to be carried by three (2.85% each) isolates. Interestingly, none of the STEC isolates belonged to serogroup O157.     

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.     

Occurrence and Characteristics of CS31A Antigen‐Producing Escherichia coli in Calves with Diarrhoea and Septicaemia in Argentina

CS31A is a K88‐related non‐fimbrial adhesin first described on Escherichia coli strains isolated from diarrhoeic and septicaemic calves. In this report, CS31A antigen was screened by immunological methods and confirmed by PCR among bovine E. coli isolates. In addition, CS31A‐producing strains were characterized with respect to different fimbrial antigens, O‐serogroup and other properties related to virulence. Faecal or tissue specimens of 100 diarrhoeic or septicaemic calves and 27 older cattle with different pathologies from 71 outbreaks or individual cases that occurred in Buenos Aires province, Argentina, were examined. CS31A+ E. coli strains were isolated from 21 (21.0%) calves from 16 outbreaks or individual cases. No CS31A+ E. coli was detected in samples from cattle more than 1 year old. Fimbriae F5, F41, F17a and F17b were not detected among the CS31A‐producing strains. Three (14.3%) of the CS31A+ E. coli strains expressed the F17c fimbria. All of the 21 isolates exhibited at least one property of septicaemic strains (resistance to serum, production of aerobactin or colicins) but none of them demonstrated heat‐stable enterotoxigenic activity. CS31A+ E. coli isolates belonged to 10 serogroups, more commonly O8, O7, O17 and O21. The results obtained here confirm the worldwide distribution of CS31A antigen in bovine E. coli strains. However, CS31A+ or CS31A+/F17c+ E. coli were less frequently isolated than they were in North hemisphere countries.     

Identification and detection of three new F17 fimbrial variants in Escherichia coli strains isolated from cattle

F17 fimbriae are produced by pathogenic Escherichia coli involved in diarrhea and septicemia outbreaks in calves and lambs. These proteins result from the expression of four different clustered genes, namely f17A, f17D, f17C and f17G, encoding a pilin protein, a periplasmic protein, an anchor protein and an adhesin protein, respectively. Several variants of f17A and f17G genes have been reported and found genetically associated with typical virulence factors of bovine pathogenic E. coli strains. In this study, a new F17e-A variant, closely related to F17b-A, was identified from a collection of 58 E. coli isolates from diarrheic calves in Iran. While highly prevalent in Iranian F17-producing clinical isolates from calves, this variant was rare among E. coli from a French healthy adult bovine population, suggesting a possible association with virulence. The f17Ae gene was also found in the genome of the Shiga-like toxin variant Stx_1d–producing bovine E. coli strain MHI813, and belonged to a gene cluster also encoding a new F17-G3 variant, which greatly differed from F17-G1 and F17-G2. This gene cluster was located on a pathogenicity island integrated in the tRNA pheV gene. The gene coding for a third new F17f-A variant corresponding to a combination of F17c-A and F17d-A was also identified on the pVir68 plasmid in the bovine pathogenic E. coli strain 6.0900. In conclusion, we identified three new F17-A and F17-G variants in cattle E. coli, which may also have significant impact on the development of new diagnostics and vaccination tools.     

Phylogenetic background and virulence genes of Escherichia coli isolates from colisepticemic and healthy broiler chickens in Iran

The purposes of this study were to determine the phylogenetic background and the virulence gene profiles of Escherichia coli isolates from colisepticemic and feces of healthy (AFEC) broiler chickens. In this study, 253 E. coli isolates including 141 avian pathogenic E. coli (APEC) and 112 AFEC isolates were examined by PCR. In general, 253 E. coli isolates distributed among group A (51.8%), B1 (15.8%), B2 (8.7%), and D (23.7%). Ten (8.9%) AFEC isolates segregated in to B1 phylo-group and 102 (91.1%) isolates fell into six different phylogenetic subgroups. Distribution of colisepticemic and fecal isolates differed significantly in their assignments to A and B1 phylo-groups. The three most prevalent virulence genes were crl, fimH, and aer in isolates between both groups. The four genetic markers aer, papC, afa, and sfa were detected significantly more often among colisepticemic isolates than in fecal isolates from healthy broilers. The presence of stx ₂ gene in fecal isolates were significantly differs among the colisepticemic isolates. F17 fimbrial family encoding gene and eae gene were detected in APEC and AFEC isolates, respectively. The colisepticemic and fecal isolates possessed the virulence genes were detected in all of the four phylogenetic groups. Several combination patterns of the virulence genes were detected in APEC and AFEC isolates. In colisepticemic isolates the combination of aer, crl, and fimH genes was the most prevalent pattern. None of the examined isolates harbored the cdt, cnf1, ipaH, and stx ₁ virulence gene sequences.     

Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia

Two hundred and fifty Escherichia coli isolates from diarrhoeic and healthy piglets were serotyped and tested for the presence of virulence genes for fimbriae, intimin, heat-labile (LT) and heat-stable (STa and STb) enterotoxins, Stx toxins, and enteroaggregative heat-stable 1 (EAST1) enterotoxin by polymerase chain reaction (PCR). Although 220 isolates from diarrhoeic piglets belonged to 43 O serogroups and 77 O:H serotypes, 60% were of one of the 10 serogroups O2, O8, O15, O54, O84, O101, O141, O147, O149 and O157, and 60% belonged to only 10 serotypes (O8:H-, O54:H-, O84:H7, O101:H-, O141:H-, O141:H4, O147:H-, O149:H10, O163:H-, and ONT:H-). PCR showed that 79% of 220 isolates carried genes for at least one of the virulence factors tested. The gene encoding for EAST1 was the most prevalent (65%) followed by those encoding for STb (49%), LT (42%), STa (13%), and Stx2e (4%). Eighty-three (38%) of the 220 E. coli isolates carried the gene for F4 (K88), whereas genes for F18, F5 (K99), F41, F6 (P987), F17, and intimin (eae) were detected in 9%, 3%, 3%, 3%, 1%, and 3%, respectively. Seropathotype O149:H10:F4:LT/STb/EAST1 (70 isolates) was the most common, representing 32% of isolates. Pulsed-field gel electrophoresis (PFGE) analysis with XbaI of 15 O149:H10 representative isolates from diarrhoeic piglets distinguished 14 types. The 15 isolates exhibited a wide variability of distinct restriction patterns though all belonged to the same serotype (O149:H10), and all but one showed identical virulence determinants (F4, LT, STb, and EAST1). Among 30 isolates from healthy piglets only two virulence genes were detected: EAST1 (26%) and eae (17%). In total, 12 isolates were positives for the eae gene: five isolates had intimin beta1, four possessed intimin theta and three showed intimin type xiB. This is believed to be the first study describing the presence of intimin type xiB in E. coli of porcine origin.     

Genetic Characterization of Antibiotic Resistance in Enteropathogenic Escherichia coli Carrying Extended‐Spectrum β‐Lactamases Recovered from Diarrhoeic Rabbits

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended‐spectrum β‐lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K‐:H2) was observed among 17 EPEC strains, the O92:K‐serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K‐serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim–sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The bla_TEM gene was demonstrated in the majority of ampicillin‐resistant isolates. The aac(3)‐II or aac(3)‐IV genes were detected in the four gentamicin‐resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin‐resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline‐resistant isolates. A total of nine EPEC isolates showed the phenotype SXT‐resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.     

Genotypic Prevalence of the Adhesin Involved in Diffuse Adherence in Escherichia coli Isolates in Pre‐weaned Pigs with Diarrhoea in Korea

A total of 1002 Escherichia coli strains isolated from pre‐weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat‐stable (STa, STb) and heat‐labile (LT) enterotoxin, enteroaggregative E. coli heat‐stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty‐three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre‐weaned pigs with diarrhoea.     

Serogroup distribution,diversity of exotoxin gene profiles,and phylogenetic grouping of CTX-M-1- producing uropathogenic Escherichia coli

The emergence of CTX-M-1 producing Uropathogenic Escherichia coli (UPEC) has become a serious challenge. In addition to antimicrobial resistance, a number of virulence factors have been shown. Therefore, this study was designed to determine the prevalence of O- serogroups, phylogenetic groups, exotoxin genes, and antimicrobial resistance properties of CTX-M-1- producing UPEC. A total of 248 UPEC isolates were collected. The antibiotic resistance was performed, and PCR was used to detect the bla_CTX-M1, exotoxins, serogroups and phylogroups of UPEC. Of 248 isolates, 95 (38.3%) harbored bla_CTX-M-1. Of them, serogroups O1 and O25 were predominant, accounting for 20% and 13.7%, respectively. The hlyA was the dominant exotoxin gene (32.6%), followed by sat (28.4%), vat (22.1%), cnf (13.7%), picU (8.4%), and cdt (2.1%). The hlyA gene was significantly associated with pyelonephritis (P = 0.003). Moreover, almost half of the isolates (45.4%) belonged to phylogenetic group B2. Most of exotoxin genes were present in significantly higher proportions in group B2 isolates except cdt gene (P < 0.05). All of the isolates were susceptible to imipenem, nitrofurantoin, and fosfomycin. The CTX-M-1-producing UPEC strains causing nosocomial infections are more likely to harbor certain exotoxin genes, raising the possibility that this increase in virulence genes may result in an increased risk of complicated UTI.     

Virulence repertoire of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic Escherichia coli (ETEC) from diarrhoeic lambs of Arunachal Pradesh,India

A total of 107 faecal samples were collected from diarrhoeic lambs of high altitude terrains (2,000 to 5,000 m above the mean sea level) of Tawang and West Kameng districts of Arunachal Pradesh, India. Total 234 Escherichia coli were isolated and further subjected to PCR for the study of virulence repertoire characteristics of Shiga toxin-producing E. coli (STEC) and enterotoxigenic E. coli (ETEC). Out of the 234 isolated E. coli, 32% were found positive for STEC, and 9% were carrying virulence gene for ETEC. The isolated STEC serogroups were O159, O127, O120, O113, O60, O30, O25, O8 and O2. Of all the 74 STEC strains, PCR showed that 18% isolates carried stx ₁ , 26% possessed stx ₂ and 47% produced positive amplicon for both. Other virulent attributes like intimin (eaeA), enterohaemolysin (ehxA) and STEC auto-agglutinating adhesin (saa) were present in 18%, 43% and 44% of the isolates, respectively. The isolated ETEC serogroups were O172, O170, O159, O146, O127, O120, O113, O86, O75, O60, O30, O25, O8, O2, OR and OUT. Of the 22 ETEC-positive isolates, 23%, 18% and 4.5% possessed the gene only for LT, STa and STb, respectively, whereas 54% carried genes for both LT and STb. Some serogroups of E. coli like O159, O127, O120, O113, O60, O30, O25, O8 and O2 possessed genes for both Shiga toxin and enterotoxin. This study is the first report of ETEC isolation from diarrhoeic lambs in India. The moderately high proportion of STEC and ETEC in the diarrhoeic lambs implicated that these animals are important reservoir of STEC and ETEC. This is really a grave concern for the ‘brokpas’ and nomads (shepherds) who share a close relationship with this animals for their livelihood. This study also indicates that ETEC may be a major cause for frequent diarrhoeal episodes in lambs of this region.     

Virulence-associated genes in avian pathogenic Escherichia coli from laying hens in Apulia,southern Italy

1. Escherichia coli isolated from lesions (Avian Pathogenic E. coli?-?APEC) of layer hens affected by colibacillosis and from intestinal contents of clinically-healthy birds (Avian Faecal E. coli?-?AFEC) were serotyped. All the isolates were investigated for the presence of virulence genes to determine which genes were more closely related to those from lesions.

2. A number of different serogroups were detected, O78 being predominant among the isolates from colibacillosis.

3. E. coli isolated from lesions were not linked to a specific pathotype (set of common virulence genes).

4. The presence of the virulence genes, with the exception of astA, was associated more generally with APEC strains.

5. Statistically, genes such as cva/cvi, tsh, iss, irp2 and iucD were more related to isolates from colibacillosis.

6. It is suggested that the detection of these genes in a rapid and inexpensive test for field practitioners could provide useful information about the potential virulence of E. coli isolated in commercial layer flocks.     

Phenotypic and genotypic properties of Escherichia coli isolated from colisepticemic cases of Japanese quail

This study was conducted to characterize the Escherichia coli isolates from colisepticemic Japanese quails. One hundred and nine E. coli were isolated in pure culture from heart blood of dead Japanese quails. The sampled birds were originated from four different farms. Antibiotic resistance pattern of E. coli isolates were determined against nine antibacterial agents. Phylotype and virulence genes of the isolates were detected by polymerase chain reaction. By disc diffusion method, all of the isolates showed resistance to three or more antibiotics, and 19 different patterns of multiple drug resistance were observed. Phylotyping of the most prevalent multiple drug-resistant isolates revealed that they mostly belonged to phylogroups A (A₁ subgroup). The E. coli isolates belong to four phylogenetic groups: A (55.0%), B1 (18.3%), B2 (17.4%), and D (9.2%). Eighty-nine (81.7%) isolates were distributed in five phylogenetic subgroups including 22 (20.2%) in A₀, 38 (34.9%) in A₁, 19 (17.4%) in B2₃, 7 (6.4%) in D₁, and 3 (2.8%) in D₂. The examined E. coli isolates exhibit at least one of the virulence genes tested, whereas three most prevalent genes were crl (94.5%), fimH (89.0%), and iutA (51.4%), respectively. The genetic marker for Afa (afaI B-C), S (sfa/focD-E), and P (papE-F) fimbriae were found in one, four, and ten isolates, respectively. Thirteen different combinations of virulence gene were observed, where combination of crl and fimH genes was the most prevalent pattern. None of the isolates contained the ipaH, stx1, stx2, and eaeA genetic markers. In conclusion, E. coli strains could be considered as a causative agent of mortality in quail farms. In conclusion, E. coli isolates from colisepticemic quails are distributed in different phylogroups, are resistant to combinations of antibiotic agents, and contain several virulence genes.     

Determination of antibiotic resistance pattern and virulence genes in Escherichia coli isolated from bovine with subclinical mastitis in southwest of Iran

The aims of the present study were to investigate the prevalence of some virulence genes and also determine the antimicrobial resistance pattern of E. coli isolated from bovine with subclinical mastitis. The milk of 502 cows was collected from 8 dairy herds in the southwest of Iran. Conventional biochemical tests were used for identification of E. coli at the species level. Antimicrobial susceptibility patterns of E. coli isolates were determined by disc agar diffusion method and polymerase chain reaction (PCR) was used for detection of seven virulence genes including f17A, afaE-8, afaD-8, eaeA, cnf1, cnf2, and iucD. Seventy (13.94%) isolates of E. coli were identified in 502 milk samples. The highest rate of resistance was observed against tetracycline (18.6%), while none of the isolates were resistant to streptomycin. Eight (11.5%) out of 70 E. coli isolates carried at least one of the virulence genes. The afaD-8 was the most prevalent gene detected in 5 (7.1%) isolates. The afaE-8, iucD, and eaeA were detected in 3, 3, and 2 isolates respectively. Low prevalence of virulence factors may be indicating that most of the E. coli isolates originated from the commensal flora of cows and enter to the udders via environment contamination with feces.

     

Characterization of Necrotoxigenic Escherichia coli (NTEC) Strains Isolated from Healthy Calves in Poland

Faecal samples from 132 healthy, 4–8‐week‐old calves from four different farms were examined for necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2). CNF2 genes were detected by polymerase chain reaction in 24 (6.1%) of the 396 E. coli strains tested; these strains were found in 18 (13.6%) calves used in the study. None of the 396 E. coli isolates examined possessed the gene encoding CNF1. Overall, 28.8% of E. coli examined expressed the F17 fimbrial antigen. A strong association between CNF2 toxin and F17 fimbriae was found (62.5% of CNF2‐positive strains were F17‐positive). Moreover, six out of 24 NTEC strains had the Stx1 or the Stx2 shiga toxin genes, and three additional isolates possessed the eae genetic marker of the intimin protein.     

Prevalence of serogroups and virulence factors of Escherichia coli strains isolated from pigs with postweaning diarrhoea in eastern China

This study was undertaken to determine the present distribution of serogroups, hemolytic activity and virulence factors among Escherichia coli strains isolated from pigs with postweaning diarrhoea from eight provinces in eastern China. Two hundred and fifteen E. coli isolates were serogrouped with O-antisera, investigated for hemolytic activity, assessed for F4, F5, F6, F18 and F41 fimbrial antigens by monoclonal antibodies and detected for genes of enterotoxins and shiga-toxin-two-variant (Stx2e) by a multiplex polymerase chain reaction (PCR). Among these E. coli isolates, 140 were determined to be placed in serogroups, 52 were unable to be serogrouped and the rest 23 auto-agglutinated. These isolates distributed in 45 serogroups and 64.3% (90/140) belonged to 12 O serogroups: O8, O9, O11, O20, O32, O91, O93, O101, O107, O115, O116 and O131. Hemolytic activity was detected in 11.6% (25/215) of all isolates. Several uncommon O serogroups were discovered in this study. Agglutination tests showed that 50.2% (108/215) of these isolates were positive for one or more of the five fimbrial antigens. Seventy-two E. coli strains expressed single fimbria and 36 strains expressed two or more fimbriae. Among these 215 E. coli isolates, strains expressing F18, F4, F6, F6 + F18 or F5 + F41 occurred more frequently. PCR analysis showed that 60.5% (130/215) of the isolates only harboured the gene of estI (STI) while 6.0% (13/215) strains possessed the genes of stx2e, estI and estII and 5.6% (12/215) of strains had the genes of estI/estII. Of all these isolates, 107 (49.8%) were negative for the fimbrial antigens examined. The fimbria-negative isolates usually possessed genetic determinant of estI (78, 72.9%).     

Serogrouping,phylotyping, and virulence genotyping of commensal and avian pathogenic Escherichia coli isolated from broilers in Hamedan,Iran

In the present study, 100 Avian-Pathogenic Escherichia coli (APEC) isolates from colibacillosis-suspected broilers and 100 Avian Faecal Escherichia coli (AFEC) isolates from healthy broilers in Iran were examined by PCR for confirmation of their serogroups and phylogenetic background, and their association with ten virulence-associated genes (VAG) including fimC, iutA, chuA, sitA, iss, cvaA/B, hylA, stx1, stx2, and yjaA. Serogroups O78, O1, O2 and O18 were the prominent strains including 54 % of the APEC and 23 % of the AFEC strains. At phylotyping, the majority of APEC strains belonged to phylogenetic group E (22 %) while for the AFEC strains, half of the isolates were not assigned to any group but the predominant phylogroup was E (27 %). Virulence genotyping, revealed that the predominant VAGs were iutA (97 %), fimC (87 %) and iss (84 %) among APEC strains, and fimC (95 %), iss (93 %) and sitA (87 %) in AFEC strains. This is the first time that phylogroup E is described as predominant phylogroup among APEC strains also, this is the first report on the presence of the stx1 gene in APEC strains isolated from broilers in Iran. The results of the present study indicate that VAGs are more prevalent in APEC strains belonging to O2 and O78 serogroups, also phylogroups E and D have more frequency of VAGs than other phylogroups. Therefore, the APEC strains belonging to O2 and O78 serogroups and phylogroups E and D probably have more pathogenicity to broilers.     

Characterization of escherichia coli isolated from adult horses with and without enteritis

Summary

In the present study E. coli strains isolated from the faeces of ten horses with diarrhoea and 14 horses without diarrhoea were characterized. All horses were culture negative for Salmonella species. Nine colonies of E. coli from each faecal sample were picked at random and a DNA fingerprint was made by means of a polymerase chain reaction (PCR) using Enterobacterial Repetitive Intergenic Consensus (ERIC) primers. The number of E. coli genotypes did not differ significantly between horses with and without diarrhoea. In addition, all E. coli strains with different DNA fingerprints were tested by PCR for genes encoding the virulence factors K88, F41, F17, CS31a, Stal, LT1, VT2, CNF, BFP, and intimin. Genes coding for K88, F41, BFP, STal, VT2, and CS31A were not detected. Genes for CNF were found in strains from one horse with diarrhoea and one horse with normal faeces. Genes for LT1 (n=1) and intimin (n=1) were found only in strains from horses with normal faeces. Genes for F17 fimbriae were found in strains from three horses with diarrhoea (30%) and in none of the strains from healthy horses. In two of these horses, E. coli strains with different DNA polymorphism patterns were F17 positive; however, none of these strains possessed LT1, Sta1, or CNF genes. Haemolytic E. coli strains were only isolated from two horses with diarrhoea and from none of the healthy horses. Nineteen percent of all E. coli strains did not ferment lactose. Eight per cent of these lactose‐negative strains were from horses with diarrhoea, whereas 32% were from horses without diarrhoea. In conclusion, virulence factors were present in E. coli isolates from horses with and without diarrhoea, except for F17, which was only found in E. coli isolated from horses with diarrhoea. F17‐positive E. coli might have importance as cause of diarrhoea in horses, but further studies are needed.     

Occurrence of genes encoding aminoglycoside-modifying enzymes in Escherichia coli isolates from chicken meat

ABSTRACT

1. The aim of the experiment was to determine the occurrence of genes encoding aminoglycoside-modifying enzymes (AMEs) in Escherichia coli isolates recovered from chicken meat.

2. Antibiotic sensitivity was tested using the disc diffusion test. AMEs and virulence profile were determined by PCR/sequencing.

3. Out of 195 meat samples collected, 185 (95%) isolates were identified as E. coli. Disc diffusion showed a resistance value of 22% (n = 42) for at least one of the antibiotic aminoglycosides (AGs) tested (tobramycin, gentamycin, amikacin and kanamycin). PCR screening showed the presence of three classes of AMEs, namely, aac(3)-II (12%), aac(6?)-Ib (7%) and aac(2?)-Ia (5%). Eight of the 42 isolates were positive for the stx₁ and sxt₂ genes and were defined as Shiga toxin-producing E coli., while the eae gene was positive in one strain. Among the 42 isolates, group A was the predominant phylogenetic identified (76%), followed by group D (21%). One isolate belonged to subgroup B2₃.

4. The results suggested that chicken meat could be an important reservoir of AMEs, and pose a potential risk by dissemination of resistance to humans through the food chain.     

Serotypes of Escherichia coli isolated from septicaemic chickens in Galicia (Northwest Spain)

The purpose of this study was to establish the serogroups of Escherichia coli that cause avian colibacillosis in Spain. The serogroups of 625 avian E. coli isolated between 1992 and 1993 were determined. The 458 E. coli from chickens with septicaemia belonged to 62 different O serogroups; however, 59% were of one of 18 serogroups (O1, O2, O5, O8, O12, O14, O15, O18, O20, O53, O78, O81, O83, O102, O103, O115, O116 and O132). These 18 serogroups were also determined as an important percentage (29%) of control isolates from faeces of healthy birds. Nevertheless, a significant difference (59% versus 29%; P<0.001) was observed. Furthermore, the serogroups O12, O14, O18, O53, O78, O81, O102, O115, O116 and O132 were almost exclusively identified among septicaemic E. coli (31% versus 3%; P<0.001). The high prevalence of O18, O81, O115, O116 and O132 isolates was not expected and may indicate the emergence of five new serogroups associated with avian colibacillosis not yet reported.     

Duplex real-time PCR assays for rapid detection of virulence genes in E. coli isolated from post-weaning pigs and calves with diarrhoea

Duplex real-time PCR assays were used as modules to cover partially automated detection of 12 genes encoding adhesins, enterotoxins and Shiga toxins in faecal E. coli isolates. For this a total of 194 E. coli isolates from pigs suffering from post-weaning diarrhoea (PWD), including 65 isolates with haemolytic activity, and 83 isolates from calves with diarrhoea were examined. Data obtained by PCR were compared with O-typing and with haemolytic activity as indirect virulence markers. E. coli O-types O139:K82, O141:K85, and O149:K91 accounted for 43.8% (n = 85) of all porcine strains and for 55.4% (n = 36) of the porcine strains, which exhibited haemolytic activity. These strains carried virulence genes by 65.9% (n = 56) and 80.6% (haemolytic E. coli, n = 29), respectively. The E. coli O-types O139:K82 and O141:K85 were significantly associated with the adhesin gene F18, and O149:K81 with the F4 gene. In this context, detection of the gene encoding F18 was coupled predominantly with the genes responsible for the production of the toxins ST-I, ST-II and Stx2, and the F4 gene with those of the enterotoxins ST-I, ST-II and LT. Both virulence patterns were detected more pronounced in E. coli strains with haemolytic activity. Fifty-six of a total of 83 E. coli isolates originating from calves were O-typed as O101 (O101:K28, O101:K30, O101:K32; n = 29), O78:K80 (n = 23), and O9:K35 (n = 4). Most of the E. coli O78:K80 strains carried the F17 gene (69.6%, n = 16). Virulence genes encoding for F4, F5 or ST-I were detected only in single cases. Intimin and Shiga toxin genes that are present in enterohaemorrhagic E. coli (EHEC) were not detected.