Detection and characterization of Shiga toxin-producing Escherichia coli in captive non-domestic mammals

Shiga toxin producing-Escherichia coli (STEC) is an important emerging pathogen, and ruminants are recognized as their main natural reservoir. The aim of this work was to establish the frequency of STEC in non-domestic mammals of the Zoo and Botanical Garden of La Plata City, Argentina, and to pheno-genotypically characterize STEC isolates. By polymerase chain reaction (PCR), Shiga toxin (stx) gene sequences were detected in 50.8% of 65 fecal samples. Twenty-five STEC strains were isolated from 38.5% of the Zoo's animals. Ten species of order Cetartiodactyla and one species of order Rodentia were recognized as new STEC carriers. STEC strains belonged to 7 different serotypes including new serotypes O12:H25 and O13:H6. Serotype O146:H28, previously associated with human infections, represented 24% of STEC isolates. The most frequent Shiga toxin identified were type 1c and type 2c. Nineteen strains were positive for iha gene, 8 strains were positive for ehxA gene. Moreover, all strains were positive for lpfAO113 and negative for rfbO157, eae, saa, lpfAO157/OI-141, lpfAO157/OI-154, efa1, and toxB genes. Results obtained by XbaI-pulsed-field gel electrophoresis (XbaI-PFGE) confirmed the transmission of STEC strains among different animal species and suborders. In addition, we observed a potential association between STEC-harboring animal and factors such as belonging to order Cetartiodactyla, living in a pit, and belonging to a non-autochthonous species. This is the first work developed with zoological mammals and STEC in Argentina.     

Characterization of Escherichia coli strains isolated from pigs with edema disease.

Escherichia coli strains belonging to serogroups O 138 and O 139 isolated from pigs with edema disease, were characterized with respect to the presence of genes encoding Shiga-like toxin I, Shiga-like toxin II and Shiga-like toxin IIv (SLT I, SLT II and SLT IIv). Genes coding for the heat-stable and heat-labile enterotoxins (ST I and LT I) were also detected. Plasmid profiling, restriction enzyme digestion of total DNA, and ribotyping were performed for further characterization of the strains. The oligonucleotide probes applied in this study appeared to be useful tools for detecting genes coding cytotoxins and enterotoxins. DNA from 12 of 16 strains hybridized with two SLT II probes, and DNA from two SLT IIv encoding strains also hybridized with the ST I probe. DNA from one SLT IIv negative strain hybridized with the LT I probe. The results from plasmid profiling, restriction enzyme digestion, and ribotyping were compared with serogrouping in attempts to distinguish between the different E. coli edema disease isolates. Fourteen different plasmid profiles were identified, and as restriction patterns barely did, and ribotyping patterns did not, reveal any information useful for differentiation of the strains beyond serogroup level, plasmid profiling seemed to be the most suitable method for discrimination between the edema disease strains investigated here.     

Virulence genes of Escherichia coli strains isolated from mastitic milk

Escherichia coli, a Gram-negative environmental pathogen associated with bovine mastitis was isolated from the milk of 34 symptomatic cows that had been diagnosed with clinical mastitis. Eighty isolates were obtained over a 17-month period and these isolates were screened by DNA amplification for the following E. coli virulence genes: cnf1, cnf2, eaeA, eagg, einv, ltx1, stx1, stx2 and vt2e. Thirty of the bacterial isolates, obtained from 23 different cows, had toxin genes identified in their DNA. The most common virulence gene detected was stx1, with a prevalence of 31%, followed by cnf2 (7.5%), vt2e (6.25%) and eaeA (4%). The possession of different virulence genes by the bacterial isolates had no discernable impact on the health status of the cows as there was no correlation between the potential for toxin production by the E. coli isolates and the systemic clinical condition of the respective infected cows.     

O-serovar distribution and antibiotic sensitivity of Pseudomonas aeruginosa strains from birds and reptiles

100 strains of Pseudomonas aeruginosa (P.a.) from birds and reptiles were compared by determination of their O-serovars and their resistance to chemotherapeutic agents. A great number of isolates (birds 17.4%, reptiles 29.6%) were serologically untypable using 17 O-antisera by slide-agglutination-technique. The prevalence of O-serovar 0:6 was found in birds (39%) and reptiles (18.5%), followed by bird-isolates 0:1 and 0:3 (each 13%) and reptile-isolates 0:16 (14.8%). The serological distribution was different among bird- and reptile-isolates and also among the human and animal strains. All strains were resistant to penicillin G and ampicillin, more than 90% to nitrofurantoin, sulfamethoxazole-trimethoprim, chloramphenicol and erythromycin. Resistance to tetracycline was found to be 87%, resistance to sulfonamide 81%, respectively. 32% of all isolates were resistant to streptomycin, 61% to kanamycin. All isolates were susceptible to genta- and neomycin. Also all isolates, except one reptile-strain, were susceptible to gyrase-blocker (Bay VP 2674). 2 isolates were resistant to polymyxin B.     

Characterization of shiga toxin-producing Escherichia coli strains isolated from calves in Poland

Fecal samples from 67 3–5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E.coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2c shiga toxin type gene. The enterohemolysin (hlyA) and intimin (eae) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans.     

Characterization of a new fimbrial antigen present in Escherichia coli strains isolated from calves.

Thirteen Escherichia coli strains isolated from calves with diarrhoea, supposed to carry a common antigen were examined for their hemagglutinating activity and compared by bacterial agglutination, double diffusion in two dimensions and by crossed immunoelectrophoresis (CIE). Two of the strains were examined also in the electron microscope. Most of the strains agglutinated red blood cells of horse, ox, guinea pig and chicken, of which the agglutination of ox erythrocytes was mannose-resistant (MRHA). None of the strains agglutinated human erythrocytes. All strains with MRHA of ox red blood cells, regardless to their O:K:H antigens could be agglutinated in unabsorbed or absorbed antisera produced against cultures C1209 (020:K-:H9) and C1213 (09:K36:H-) when live cells as antigens were used. None of these sera agglutinated reference strains carrying K88, K99, 987P, F41 or FY (Att25) antigen respectively. By the double gel diffusion test and by CIE in extracts (60 degrees C) of the strains a common heat labile antigen, responsible for the MRHA of ox red blood cells was identified. Electron microscopy revealed that this common antigen was represented by thin, long, hair-like fimbriae on cells of E. coli C1213, and that specific homologous antibodies attached to these fimbriae.     

Cytotoxins in non-enterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves

We have examined the cytotoxic responses produced in HeLa and Vero cell cultures by sonicates from 15 non-enterotoxigenic (STa-, LT-) strains of E. coli, highly lethal for mice parenterally LD50 less than 3 X 10(7) CFU), which had been isolated from feces of diarrheic calves. Three types of cytotoxic responses were observed. Type 1 (five strains) consisted of enlargement, rounding and polynucleation of HeLa cells, an effect previously reported with cytotoxic necrotizing factor (CNF) in E. coli from infant and piglet enteritis. Type 2 toxicity (three strains and the control Vir strain S5) was also characterized by enlargement and polynucleation of HeLa cells, but in contrast to Type 2 effect, cells were elongated. Sonicates from the latter strains were lethal for chickens, producing the lesions previously described with Vir strains. Type 3 toxicity (two strains and the control VT strain H19), produced an extensive destruction of both Vero and HeLa cell cultures. Cytotoxic effects were completely abolished upon heating for 1 h at 60 degrees C for Type 1 and 2 extracts and at 80 degrees C for Type 3 extracts. Seroneutralization assays showed that cytotoxins of the same type were closely related antigenically. In addition, a slight cross-neutralization was observed between Type 1 (CNF) and Type 2 (Vir) toxins.     

Antibiotic resistance of Escherichia coli strains isolated from chickens with colisepticaemia in Morocco

Sixty-two strains of Escherichia coli were isolated in 58 farms from broiler chickens showing respiratory signs and lesions characteristic of avian colibacillosis. Serological examination of these strains showed that the types 078, 01 and 02 (for the somatic antigen) and K1 (for the capsular antigen) were the most frequently found. Newcastle disease virus was also isolated in two cases. All the strains of E. coli isolated were sensitive to colistin, flumequine and gentamicin. A few strains were resistant to neomycin, nalidixic acid and trimethoprim. The frequency of strains resistant to nitrofurans, sulfonamides, chloramphenicol, spectinomycin and ampicillin was intermediate. Most strains were resistant to tetracycline. Multiple resistance was common.     

Isolation and identification of mycobacteria from captive reptiles

The occurrence of Mycobacterium species in clinically healthy pet reptiles was studied in Italy during the period 2004-2006. The feces samples of 223 animals were examined bacteriologically. Thirty-seven strains were isolated, in particular from 13/18 (72.2%) ophidians, 13/134 (9.7%) saurians and 11/71 (15.5%) chelonians. The isolates were classified, after HPLC analysis of bromophenacyl esters of cell wall mycolic acids, as Mycobacterium fortuitum (14 strains, 37.8%), Mycobacterium fortuitum-like (17, 45.9%), Mycobacterium peregrinum (4, 10.8%), and Mycobacterium chelonae (1, 2.7%). M. fortuitum was isolated from seven pythons, five saurians and two turtles; M. fortuitum-like from six saurians, six pythons and five turtles; M. peregrinum from four turtles; M. chelonae from one lizard. One isolate from an Iguana iguana could not be identified by HPLC analysis showing a previously unreported profile. Comparative 16S rDNA sequencing showed a low similarity with Mycobacterium triviale (97.2%) and Mycobacterium confluentis (97.1%). On the basis of such data the unidentified bacterium turned out to belong to a not yet described Mycobacterium species.     

Characteristics of alpha-hemolytic strains of Escherichia coli isolated from dogs with gastroenteritis

Twenty-four hemolysin producing (Hly+) strains of Escherichia coli isolated from dogs with gastroenteritis were investigated for their virulence markers and their phenotypic properties. The strains were distributed over eleven known E. coli O-serogroups and most of them were heterogeneous for their phenotypes. All strains were found to produce alpha-hemolysin which was detected by Southern hybridization and colony immunoblotting using a specific gene probe and a monoclonal antibody. Eight strains were carrying plasmids encoding alpha-hemolysin sequences (hly-plasmids) and 16 strains carried chromosomal hly-determinants. Twelve of the strains showed enterotoxic activities which were tested for in different assays. Among these, three O42:H37 and two O70:H-strains carrying hly-plasmids were found to harbour other plasmids encoding the heat-stable enterotoxin STA1. The other seven strains showing enterotoxicity in the ileal loop or the suckling mouse assay were negative for STA1, STA2, or LT. None of the 24 strains were positive for invasiveness or for production of Vero (Shiga-like) toxins. The production of alpha-hemolysin was closely associated with the production of cytotoxic necrotizing factor (CNF), which was detected in 17 of 24 strains. Of these, 16 elaborated CNF1 and one strain produced an unknown CNF type. Surprisingly, all strains carrying ST-plasmids and six of eight strains carrying hly-plasmids were negative for CNF. Thus, in canine E. coli strains CNF production seems to be closely associated with production of chromosomally encoded alpha-hemolysin whereas hly-plasmids are more often associated with ST-producing, CNF negative isolates.     

Drug resistance in strains of Escherichia coli isolated from the intestinal tract of pigs in Norway.

Faecal samples from 95 healthy pigs and samples of jejunal content from 85 piglets suffering from colienterotoxaemia were tested for the presence of drug resistant E. coli strains. Practically all pigs in both groups harboured E. coli strains resistant to one or more of the 6 antibiotics/chemotherapeutic agents tested (Oxytetracycline, streptomycin, sulphaisodimidin, neomycin, ampicillin, chloramphenicol). Almost 100% of healthy and approx. 90% of diseased pigs harboured strains resistant to Oxytetracycline, streptomycin and sulphaisodimidin. Pigs with strains resistant to neomycin, ampicillin and chloramphenicol were less frequently found. The predominant coliform flora consisted of E. coli strains” resistant to Oxytetracycline, streptomycin and sulphaisodimidin in 71% to 81% of diseased pigs and in 47% to 69% of the healthy pigs. In diseased pigs ¾ of the animals had a coliform flora dominated by neomycinresistant E. coli strains.Of the 721 resistant E. coli strains isolated from healthy pigs, 11% were single resistant while the corresponding figure for the 518 resistant strains isolated from diseased pigs was 6%. Thus 89% and 94% of strains showed simultaneous resistance to 2 or more antibiotics. E. coli strains resistant to 3 or more drugs were found in approx. 60% and 70% of the isolates from healthy and diseased animals, respectively. Oxytetracycline/streptomycin/sulphaisodimidin resistance was most commonly found, approx. 22% and 38% of the strains from healthy and diseased pigs, respectively, showing this resistance pattern.Transmission of drug resistance which was examined in E. coli strains originating from the diseased pigs was demonstrated in approx. 76% of the isolates. The incidence of drug resistance transfer in single, double, triple and quadruple resistant strains was 11%, 68%, 97% and 98%, respectively.     

In vitro inhibition of Salmonella organisms isolated from reptiles by an inactivated culture of microcin-producing Escherichia coli

OBJECTIVE: To determine whether an inactivated culture of a microcin-producing avian Escherichia coli was capable of killing Salmonella isolates from reptiles in an in vitro test system. SAMPLE POPULATION: 57 Salmonella isolate from reptiles. PROCEDURE: A wild-type avian E. coli electrotransformed with a plasmid coding for the production of microcin 24 was tested in an in vitro microassay system for its ability to kill 57 Salmonella spp isolated from reptiles. The reptile population included snakes, iguana, frilled lizards, turtles, other lizards, and unspecified reptiles. RESULTS: 44 of the Salmonella isolates were inhibited strongly, compared with the in vitro assay controls; 12 had weak inhibition, and 1 was not inhibited by the microcin-producing E. coli. Thirteen of the 57 isolates had resistance to at least 1 antibiotic, primarily streptomycin. There were 9 O serogroups identified in the 57 isolates, with serogroup H being the most prevalent (18 to 57). CONCLUSION AND CLINICAL RELEVANCE: Antibiotics are not recommended to eliminate Salmonella organisms from reptiles because of the development of antibiotic resistance. Further studies are necessary to determine whether the use of microcin-producing bacteria will be effective in controlling Salmonella infections in companion reptiles.     

Characterization of integrons-mediated antimicrobial resistance among Escherichia coli strains isolated from bovine mastitis

To assess the prevalence of antimicrobial resistance and class I integrons in Escherichia coli strains (n=58) isolated from bovine mastitis in Inner Mongolia, antimicrobial susceptibility and the presence of various types of integrons were characterized. Most isolates were susceptible to amikacin, colistin, ceftazidime, gentamicin and kanamycin, while those also exhibited high resistant incidence rates to ampicillin, amoxicillin, sulfadiazine and sulfamethoxydiazine. The integrase gene of integrons was amplified by PCR using degenerate primers. The integrons were confirmed by restriction fragment length polymorphism (RFLP) analysis of positive PCR products. Neither class II nor class III integron was detected, while 56.90% (n=33) of the isolates were positive for the presence of intI1 gene. Sequencing analysis of gene cassettes revealed that seven gene cassettes were found, which encoded resistance to trimethoprim (dfrA1 and dfrA17), aminoglycosides (aacA4, aadA1 and aadA5) and chloramphenicol (catB3), respectively. Of them, the gene cassette array dfrA17-aadA5 was found most prevalent (62.96%). The percentage of positive-integron among the isolates whose resistant profile was relatively broad (n> or =7) is 100.00%, while the one in narrow-profile isolates (n=2-6) is 30.56%. The correlation analysis revealed the incidence of integrons among the isolates were highly related to the resistant profile, indicating integrons play an important role in the dissemination and spread of the antimicrobial resistant strains.     

Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil

Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0-11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes. These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41. Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones. The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones. Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources. Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.     

A longitudinal study of verotoxin-producing Escherichia coli in two dairy goat herds

A longitudinal study was conducted on two dairy farms to investigate the pattern of shedding of verotoxin-producing Escherichia coli (VTEC) in goats. Faecal samples were taken from 20 goat kids once weekly during the first 4 weeks of life and then once every month for the next 5 months of life, and from 18 replacement animals and 15 adults once every month for 12 months. The proportion of samples containing VTEC was higher for replacement animals and adults (85.7% and 78.7%, respectively) than for goat kids (25.4%). About 90% of the VTEC colonies isolated from healthy goats belonged to five serogroups (O33, O76, O126, O146 and O166) but the most frequent serogroups of these isolates, except one, were different in the two herds studied. E. coli O157:H7 was found in three goat kids on only one occasion. None of the VTEC isolates, except the three E. coli O157:H7 isolates, was eae-positive. The patterns of shedding of VTEC in goat kids were variable, but, in contrast, most of the replacement animals and adults were persistent VTEC shedders. Our results show that isolates of VTEC O33, O76, O126, O146 and O166 are adapted for colonising the intestine of goats but that, in contrast, infection with VTEC O157:H7 in goats seems to be transient.     

Characterization of fluoroquinolone resistance in Escherichia coli strains from ruminants.

Thirty-seven fluoroquinolone-resistant Escherichia coli strains from ruminants (according to Clinical and Laboratory Standards Institute guidelines) were screened by molecular methods for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes and for the presence of the qnrA gene. One of the strains studied was an enterohemorrhagic E. coli (EHEC) strain potentially pathogenic for humans. Three E. coli strains resistant to enrofloxacin (minimal inhibitory concentration [MIC] = 2 microg/ml) but not to ciprofloxacin (MIC = 1 microg/ml) presented single mutations in the gyrA and parC genes, while 34 strains resistant to both fluoroquinolones presented double and single mutations in gyrA and parC, respectively (31 strains), or double mutations in gyrA and parC (3 strains). The EHEC strain presented a double amino acid substitution in the GyrA protein (Ser-83-->Leu and Asp-87-->Gly) and a double amino acid substitution in the ParC protein (Gly-78-->Cys and Ser-80-->Arg), one of which has not been previously described. The present study shows that most of the mutations in the QRDR of the gyrA and parC genes of fluoroquinolone-resistant E. coli strains from ruminants are the same as those seen in E. coli strains from other animal species and humans and that there are no differences in mutation patterns in the QRDR of E. coli strains from healthy ruminants and those with diarrhea. No strains carried qnrA, which indicates that this gene does not play an important role in the selection of fluoroquinolone-resistant E. coli strains from ruminants.     

Virulence factors and genetic relatedness of Escherichia coli strains isolated from pigs with post-weaning diarrhea

Forty-six Escherichia coli strains isolated from post-weaning diarrhea of pigs were analysed for their phenotypic and genotypic properties. The isolates were of serogroups O138, O139, and O141 and most of them possessed hemolytic activities. PCR analysis showed that 34 of the isolates harboured the genes for shiga toxin 2e and 32 strains possessed the genes for heat-stable enterotoxins I and II. Ten strains had the fedA gene of F18 fimbriae. The genetic relationships among all isolates were tested by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) PCR analyses. Using the RAPD test with two different primers, six fingerprints were distinguished whereas the ERIC analysis revealed only three DNA patterns. Some strains possessing identical phenotypic and genotypic virulence determinants exhibited distinct RAPD profiles and some isolates with different pathogenic markers showed the same RAPD and ERIC pictures. Thus, RAPD, and to a less extent ERIC techniques, revealed intra- and interserogroup genotypic variations among the E. coli strains analyzed.