Escherichia coli-producing extended-spectrum beta-lactamase CTX-M-15 in a captive South American tapir (Tapirus terrestris).

Only a few reports exist on the occurrence of resistant bacteria in zoo animals. Therefore, an isolation of multiresistant Escherichia coli from the lungs of a captive South American tapir (Tapirus terrestris) lead to its characterization and further investigation of samples from animals inhabiting the same paddock and from the shared environment. The tapir suffered from an intermandibular abscess and pneumonia and was euthanatized after unsuccessful therapy, including administration of antibiotics. The authors performed selective isolation of extended-spectrum beta-lactamase (ESBL)-positive E. coli strains and identification of resistance genes using polymerase chain reaction. Seven multiresistant, ESBL-producing E. coli isolates were obtained, all belonging to the B2 phylogenetic group and showing identical profile on pulsed-field gel electrophoresis. These isolates carried several resistance genes, including the gene bla(CTX-M-15). This case demonstrates the transmission of related epidemiologically important E. coli isolates whose potential transmission to other animals and zoo staff can be assumed.

Broilers as a source of quinolone-resistant and extraintestinal pathogenic Escherichia coli in the Czech Republic.

Extraintestinal Escherichia coli infections are associated with extraintestinal pathogenic E. coli (ExPEC) strains. A total of 114 E. coli isolates were characterized regarding their antimicrobial resistance in a prospective study of 319 broilers from 12 slaughterhouses in the Czech Republic, a European Union member, during 2008. PCR-based assays to define ExPEC-associated traits were performed in resistant strains. Consumption of antimicrobial drugs by poultry in the Czech Republic was also analyzed. Antibiotic resistance was detected in 82% of isolates. Resistance to nalidixic acid and ciprofloxacin was predominant. Plasmid-mediated quinolone resistance genes, qnrB19 and qnrS1, were detected in 1 and 3 of 93 resistant isolates, respectively. Twenty-three percent of resistant isolates were considered as ExPEC. In total, 972 kg of flumequine, enrofloxacin, and difloxacin were used in poultry in the Czech Republic during 2008. High prevalence of broilers with ciprofloxacin-resistant E. coli isolates was linked to consumption of quinolones in poultry. Broilers may comprise an important vehicle for community-wide dissemination of fluoroquinolone-resistant E. coli and ExPEC. Withdrawal of fluoroquinolones from use in chicken production should be seriously considered in the Czech Republic and the European Union as well.

Experimental infection of gnotobiotic piglets with Escherichia coli strains positive for EAST1 and AIDA.

The virulence factors EAST1 and AIDA are often detected in ETEC/VTEC strains isolated from pigs and their role in diarrhoeal infections is discussed. In order to elucidate the pathogenesis of AIDA, the colonisation patterns of F4 positive and AIDA positive strains were investigated. Two wild-type Escherichia coli strains AIDA/EAST1 and F4/EAST1 isolated from diarrhoeal piglets were used for animal experiment to evaluate the ability of the EAST1 toxin to be involved in induction of diarrhoea. Gnotobiotic piglets were supplemented with normal porcine serum and orally inoculated with the strains. Faecal bacterial shedding of the challenge strains was observed during the experiment. Light microscopy and scanning electron microscopy were used to detect the colonisation pattern of both challenge strains. Although bacterial isolation demonstrated shedding of the challenge strains until the end of the experiment, diarrhoea did not develop in any piglet. Based on histological examination, piglets were more heavily colonised in the case of infection with E. coli O149/F4/EAST1 strain. Scanning electron microscopy showed bacterial cells of F4/EAST1 E. coli adhering to enterocytes, in contrast to AIDA/EAST1 which were poorly present on the intestinal surface. The EAST1 toxin alone was not able to induce diarrhoea in animals. Therefore our results demonstrate that the function/role of EAST1 and AIDA in colibacillosis of pigs remains to be elucidated.

Interaction of porcine neutrophils with different strains of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is one of the most important causes of post-weaning diarrhea in piglets. Whilst serotype O149:F4 is frequently associated with hemorrhagic gastroenteritis, other serotypes have been found to be associated with mild or moderate enteritis. As neutrophils are recruited to sites of inflammation, the aim of this study was to ascertain whether or not there is any difference in the in vitro interaction between neutrophils and two different ETEC serotypes: O149:F4 and O147:F18. The association of bacteria with neutrophils was evaluated by flow cytometry. The respiratory burst was measured by the fluorescent probe dichlorofluorescein diacetate using flow cytometry and by L012-amplified chemiluminescence. The titers of antibodies against ETEC present in cultivation sera were assessed by agglutination. The viability of E. coli was ascertained by cultivation. It was found that the strains of O149 serotype were more frequently associated with neutrophils and induced a more intensive respiratory burst compared to the strains of O147 serotype. These differences might be due to the presence of different types of fimbriae on the surface of the strains tested and by the presence of anti-fimbrial antibodies in the porcine plasma. However, the intensive interaction between E. coli and the neutrophils and respiratory burst induced by the O149 strain did not lead to more efficient killing of the bacteria. It is suggested that a stronger respiratory burst may be an important factor causing severe clinical signs of post-weaning diarrhea in piglets.

Detection of virulence factors of Escherichia coli focused on prevalence of EAST1 toxin in stool of diarrheic and non-diarrheic piglets and presence of adhesion involving virulence factors in astA positive strains.

Between 2005 and 2009, a total of 800 Escherichia coli strains isolated from piglets with diarrhea were tested for the presence of enteroaggregative heat-stable enterotoxin EAST1, heat-labile (LT) and heat-stable enterotoxins (STa) and shigatoxin (Stx2e) by PCR with the purpose of investigating the present distribution of virulence factors on swine farms in the Czech Republic. The isolates were analyzed for their O-serogroup, fimbrial (K88, K99, 987P, F41, F18) and nonfimbrial adhesins (adhesin involved in diffuse adherence AIDA and porcine attaching and effacing-associated factor PAA). The detection rates of ETEC and STEC isolates were 36.5% and 7.75%, respectively, which implies that ETEC play the major role in E. coli infections in Czech herds. Generally, the most common serotype was O149:K88 which possessed genetic determinants for LT and EAST1. None of the tested E. coli isolates was positive for genes K99, 987P and F41. It was shown that out of 800 E. coli strains isolated from pigs, 277 were EAST1 positive and 74% from the latter were identified as ETEC. Of the fimbrial adhesins, K88 and F18 were commonly detected. Over 80% of K88/EAST1 positive strains possessed the gene for paa. We detected no EAE isolate positive for fimbrial adhesins or PAA and AIDA. The AIDA was more often associated with F18 than with K88. The gene astA was also identified among E. coli isolates of non-diarrheic piglets. We tested rectal swab samples collected from apparently healthy piglets on three farms. On all farms, E. coli astA positive strains (26.66%, 90.00% and 46.66% astA positive animals) were isolated. Our results showed a significantly higher prevalence of astA positive E. coli isolates among apparently healthy piglets in comparison with diarrheic piglets. The question remains as to what is the role of the astA gene in the pathogenesis of porcine colibacillosis and as a virulence factor.

Cytokine mRNA expression in porcine cell lines stimulated by enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is an extracellular bacterium that causes post-weaning diarrhoea (PWD) in piglets with different severity of clinical signs. The pathogenesis of ETEC is ascribed to the effect of enterotoxins. ETEC colonizes ileum and probably can penetrate the epithelium and stimulate macrophages. The aim of study was to examine whether there is any difference in cytokine response in vitro produced by two porcine cell lines, intestinal epithelial cell line (IPI-2I) and macrophage cell line (3D4/31) after stimulation with different serotypes of ETEC associated with different clinical course of PWD in piglets. Three serotypes, O149:K88 (F4), O147:F18 and O8:K88, were used. We observed that all the used serotypes were unable to induce IL-8 and TNF-alpha mRNA expression in IPI-2I cell line as measured by the real-time RT-PCR. In 3D4/31 cell line, we detected differences in cytokine response among the used serotypes. The highest IL-8 and TNF-alpha mRNA expression in 3D4/31 was detected after stimulation with serotype O149:K88 frequently associated with hemorrhagic gastroenteritis.

Experimental study of pathogenicity of Pasteurella multocida serogroup F in rabbits.

The role of Pasteurella multocida serogroup F in inducing disease in rabbits was investigated in this study. Three groups of 12 Pasteurella-free rabbits each were intranasally (i.n.), subcutaneously (s.c.), and perorally (p.o.) challenged, respectively. Six rabbits of each group were immunosuppressed using dexamethasone. Eight rabbits (four of them immunosuppressed) inoculated i.n. showed symptoms of respiratory distress resulting in respiratory failure and died or were euthanized in the terminal stage of the disease 3-6 days post-infection (p.i.). The main pathological findings were fibrinopurulent pleuropneumonia (immunocompetent rabbits) or diffuse haemorrhagic pneumonia (immunosuppressed rabbits). Septicemic syndrome ending with shock occurred in 11 rabbits (6 of them immunosuppressed) inoculated s.c., which died or were euthanized in the terminal stage of the disease 2-3 days p.i. The most significant pathological findings were extensive cutaneous and subcutaneous lesions. All of the p.o. inoculated rabbits survived the challenge showing no clinical signs of the disease and no macroscopic lesions. The observations in this study indicate that in addition to serogroups A and D of P. multocida, serogroup F also can be highly pathogenic for rabbits and therefore might be a cause of considerable economic loss in commercial rabbit production.

The effect of intramuscular administration of colistin on the development and course of experimentally induced oedema disease in weaned piglets.

Shiga-toxigenic E. coli (STEC) strains that produce Shiga toxin Stx2e cause oedema disease in weaned piglets. The purpose of the present study was to investigate the impact of Stx2e released in mesenteric lymph nodes on disease pathogenesis. Colistin and ampicillin were intramuscularly administered to piglets of the experimental group simultaneously challenged with STEC strain, type O139:F18ab, Stx2e+. Piglets of the control group were challenged with STEC only. The strain was naturally resistant to ampicillin and susceptible to colistin. After the challenge, colonisation of the intestines was observed in both antibiotic-treated piglets and control piglets without antibiotic treatment. Histochemistry and scanning electron microscopy revealed sporadic colonisation of the small intestine in the piglets. STEC was detected in the mesenteric lymph nodes of untreated piglets. The clinical manifestations of oedema disease were observed in both groups. In the antibiotic-treated group (11 piglets), oedema disease developed in 10 piglets, eight of which died or were euthanized ante finem. In the untreated group (11 piglets), oedema disease developed in five piglets, four of which died or were euthanized ante finem. We therefore propose that the STEC lysed by colistin suddenly released the toxin from bacterial cells immediately after their passage through the intestinal wall. That could explain a more severe course of oedema disease in the treated piglets. Even though high amounts of STEC were present in the lymph nodes of untreated piglets, the toxin was not released abruptly because the bacterial cells were not damaged.

HlyA knock out yields a safer Escherichia coli A0 34/86 variant with unaffected colonization capacity in piglets.

Escherichia coli A0 34/86 (O83:K24:H31) has been successfully used for prophylactic and therapeutic intestinal colonization of premature and newborn infants, with the aim of preventing nosocomial infections. Although E. coli A0 34/86 was described as a nonpathogenic commensal, partial sequencing revealed that its genome harbours gene clusters highly homologous to virulence determinants of different types of E. coli, including closely linked genes of the alpha-haemolysin operon (hlyCABD) and for the cytotoxic necrotizing factor (cnf1). A haemolysin-deficient mutant (Delta hlyA) of E. coli A0 34/86 was generated and its colonization capacity was determined. The results show that a single dose of the A0 34/86 wild-type or Delta hlyA strains resulted in efficient intestinal colonization of newborn conventional piglets, and that this was still considerable after several weeks. No difference was observed between the wild-type and the mutant strains, showing that haemolysin expression does not contribute to intestinal colonization capacity of E. coli A0 34/86. Safety experiments revealed that survival of colostrum-deprived gnotobiotic newborn piglets was substantially higher upon colonization by the nonhaemolytic strain than following inoculation by its wild-type ancestor. We suggest that the E. coli A0 34/86 Delta hlyA mutant may represent a safer prophylactic and/or immunomodulatory tool with unaffected colonization capacity.

Predominance of afr2 and ral fimbrial genes related to those encoding the K88 and CS31A fimbrial adhesins in enteropathogenic Escherichia coli isolates from rabbits with postweaning diarrhea in Central Europe.

PCR tests designed in these studies identified three rabbit adhesive factor genes among 43 enteropathogenic E. coli (EPEC) strains: afr1 (2 strains), the F4(K88)/CS31A-related afr2 (10 strains), and ral (15 strains). Several EPEC strains (i.e., O153:H7 and O157:H2) lacked these genes but did adhere to HeLa cells and produced attaching and effacing lesions in rabbits.

Infectious agent and immune response characteristics of chronic enterocolitis in captive rhesus macaques.

Chronic enterocolitis is the leading cause of morbidity in colonies of captive rhesus macaques (Macaca mulatta). This study's aim was to identify the common enteric pathogens frequently associated with chronic enterocolitis in normal, immunocompetent rhesus monkeys and to elucidate the influence of this clinical syndrome on the host immune system. We analyzed the fecal specimens from 100 rhesus macaques with or without clinical symptoms of chronic diarrhea. Retrospective analysis revealed an increased incidence of Campylobacter spp. (Campylobacter coli and Campylobacter jejuni), Shigella flexneri, Yersinia enterocolitica, adenovirus, and Strongyloides fulleborni in samples collected from animals with chronic diarrhea (P < 0.05). The presence of additional enteric pathogens, such as Escherichia coli, carrying the eaeA intimin or Stx2c Shiga toxin virulence genes, Balantidium coli, Giardia lamblia, Enterocytozoon bieneusi, and Trichuris trichiura was found in all animals regardless of whether diarrhea was present. In addition, the upregulation of interleukin-1 alpha (IL-1 alpha), IL-3, and tumor necrosis factor alpha cytokine genes, accompanied by an increased presence of activated (CD4(+) CD69(+)) T lymphocytes was found in gut-associated lymphoid tissues collected from animals with chronic enterocolitis and diarrhea in comparison with clinically healthy controls (P < 0.05). These data indicate that chronic enterocolitis and diarrhea are associated, in part, with a variety of enteric pathogens and highlight the importance of defining the microbiological status of nonhuman primates used for infectious disease studies. The data also suggest that chronic colitis in rhesus macaques may have potential as a model of inflammatory bowel disease in humans.