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.     

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.     

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

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

Antimicrobial resistant bacteria in wild mammals and birds: a coincidence or cause for concern?

Background

The emergence and dissemination of antimicrobial resistance (AMR) is a growing concern to public and animal health. The contribution attributable to wildlife remains unclear. In this study two unrelated wildlife species herring gulls (Larus argentatus) and a hybrid deer (Cervus elaphus x Cervus nippon) were investigated for the presence of Escherichia coli expressing an AMR phenotype.

Findings

Bacterial isolates resistant to β-lactam compounds were identified in both animal species and the production of functional β-lactamase was confirmed using nitrocefin. The prevalence of resistant isolates was higher in herring gulls (87%) compared to deer (31%). Resistance to this class of antibiotic was found only in non-pathogenic E. coli in herring gulls and in both pathogenic and non-pathogenic E. coli strains in deer.

Conclusions

The presence of AMR in wildlife has implications for public health, food safety and potable water source protection among others.     

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

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

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

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

Changes in various metabolic parameters in blood and milk during experimental Escherichia coli mastitis for primiparous Holstein dairy cows during early lactation

Background

The objective of this study was to characterize the changes in various metabolic parameters in blood and milk during IMI challenge with Escherichia coli (E. coli) for dairy cows during early lactation. Thirty, healthy primiparous Holstein cows were infused (h = 0) with ~20-40 cfu of live E. coli into one front mammary quarter at ~4-6 wk in lactation. Daily feed intake and milk yield were recorded. At –12, 0, 3, 6, 12, 18, 24, 36, 48, 60, 72, 96, 108, 120, 132, 144, 156, 168, 180 and 192 h relative to challenge rectal temperatures were recorded and quarter foremilk was collected for analysis of shedding of E. coli. Composite milk samples were collected at -180, -132, -84, -36, -12, 12, 24, 36, 48, 60, 72, 84, 96, 132 and 180 h relative to challenge (h = 0) and analyzed for lactate dehydrogenase (LDH), somatic cell count, fat, protein, lactose, citrate, beta-hydroxybutyrate (BHBA), free glucose (fglu), and glucose-6-phosphate (G6P). Blood was collected at -12, 0, 3, 6, 12, 18, 24, 36, 60, 72, 84, 132 and 180 h relative to challenge and analyzed for plasma non-esterified fatty acids (NEFA), BHBA and glucose concentration. A generalized linear mixed model was used to determine the effect of IMI challenge on metabolic responses of cows during early lactation.

Results

By 12 h, E. coli was recovered from challenged quarters and shedding continued through 72 h. Rectal temperature peaked by 12 h post-challenge and returned to pre-challenge values by 36 h post-IMI challenge. Daily feed intake and milk yield decreased (P <0.05) by 1 and 2 d, respectively, after mastitis challenge. Plasma BHBA decreased (12 h; P <0.05) from 0.96 ± 1.1 at 0 h to 0.57 ± 0.64 mmol/L by 18 h whereas concentration of plasma NEFA (18 h) and glucose (24 h) were significantly greater, 11 and 27%, respectively, after challenge. In milk, fglu, lactose, citrate, fat and protein yield were lower whereas yield of BHBA and G6P were higher after challenge when compared to pre-challenge values.

Conclusions

Changes in metabolites in blood and milk were most likely associated with drops in feed intake and milk yield. However, the early rise in plasma NEFA may also signify enhanced adipose tissue lipolysis. Lower concentrations of plasma BHBA may be attributed to an increase transfer into milk after IMI. Decreases in both milk lactose yield and % after challenge may be partly attributed to reduced conversion of fglu to lactose. Rises in G6P yield and concentration in milk after challenge (24 h) may signify increased conversion of fglu to G6P. Results identify changes in various metabolic parameters in blood and milk after IMI challenge with E. coli in dairy cows that may partly explain the partitioning of nutrients and changes in milk components after IMI for cows during early lactation.     

Modelling the dynamics of intramammary E. coli infections in dairy cows: understanding mechanisms that distinguish transient from persistent infections

The majority of intramammary infections with Escherichia coli in dairy cows result in transient infections with duration of about 10 days or less, although more persistent infections (2 months or longer) have been identified. We apply a mathematical model to explore the role of an intracellular mammary epithelial cell reservoir in the dynamics of infection. We included biological knowledge of the bovine immune response and known characteristics of the bacterial population in both transient and persistent infections. The results indicate that varying the survival duration of the intracellular reservoir reproduces the data for both transient and persistent infections. Survival in an intracellular reservoir is the most likely mechanism that ensures persistence of E. coli infections in mammary glands. Knowledge of the pathogenesis of persistent infections is essential to develop preventive and treatment programmes for these important infections in dairy cows.     

Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.     

Genomic Description of Antibiotic Resistance in Escherichia coli and Enterococci Isolates from Healthy Lusitano Horses

Antibiotic resistance is a global problem, and it is known that commensal bacteria can act as reservoir of antibiotic resistance genes of clinical importance. The aim of the present study was to determine the antibiotic resistance phenotype and mechanisms implicated in resistance of Escherichia coli and Enterococcus spp. isolates collected from fecal samples of 90 Lusitano horses from Portugal. Sixteen of the 71 E. coli isolates (22.5%) recovered showed resistance to at least one of the antibiotics tested. The number of E. coli isolates resistant to streptomycin, tetracycline, chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, and gentamicin was 9, 7, 6, 3, 2, and 1, respectively. The bla_TEM-1 and bla_OXA-1 genes were detected in ampicillin-resistant isolates and the sul2 and dfrA1 genes in trimethoprim-sulfamethoxazole-resistant, while the aac(3)-I, floR and tet(A) were found in the gentamicin, chloramphenicol and tetracycline-resistant isolates, respectively. Twenty-two of the 71 (31%) recovered enterococci showed antibiotic resistance for at least one of the tested antibiotics, and resistant isolates were identified as Enterococcus faecium (n = 14), E. faecalis (n = 3), E. hirae (n = 2), and Enterococcus spp. (n = 3). The erm(B) and erm(C) genes were identified in erythromycin-resistant enterococci and the tet(M) and/or tet(L) genes in tetracycline-resistant isolates. The slight prevalence of antibiotic resistance among commensal bacteria of healthy Lusitano horses can improve the treatment of upcoming infections in these horses because these microorganisms can be considered as antimicrobial indicator bacteria.     

Low numbers of intestinal Shiga toxin-producing E. coli correlate with a poor prognosis in sheep infected with bovine leukemia virus

Healthy ruminants carry intestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stx has antiviral activities in vitro and STEC numbers correlate with reduced early viremia in sheep experimentally infected with bovine leukemia virus (BLV). This study assessed the impact of intestinal STEC on BLV-induced disease for one year post-BLV-challenge. High STEC scores (CFU/g feces × frequency of STEC-positive samples) correlated with good health, whereas poor weight gain, distress, and tumor development occurred only among animals with low STEC scores. STEC carriage was associated with increased percentages of B cells in peripheral blood.