Identification of Escherichia coli isolated from flies (Insecta: Diptera) that inhabit the environment of dairy farms harboring extraintestinal virulence markers.

AIMS: We investigate extraintestinal pathogenic genes (ExPEC) related to virulence of Escherichia coli in flies from the dairy environment. METHODS AND RESULTS: We collected 217 flies from nine dairy farms, which were submitted to microbiological culture. Fifty-one E. coli were identified using mass spectrometry. Eleven dipteran families were identified, with a predominance of Muscidae, and a minor frequency of Tachinidae, Drosophilidae, Sphaeroceridae, Ulidiidae, Syrphidae, Chloropidae, Calliphoridae, Sarcophagidae, and Piophilidae. A panel of 16 virulence-encoding genes related to ExPEC infections were investigated, which revealed predominance of serum resistance (traT, 31/51 = 60.8%; ompT, 29/51 = 56.9%), iron uptake (irp2, 17/51 = 33.3%, iucD 11/51 = 21.6%), and adhesins (papC, 6/51 = 11.8%; papA, 5/51 = 9.8%). CONCLUSIONS: Our findings reveal Dipterans from milking environment carrying ExPEC virulence-encoding genes also identified in clinical bovine E. coli-induced infections.

Characterization of mammary pathogenic Escherichia coli reveals the diversity of Escherichia coli isolates associated with bovine clinical mastitis in Brazil.

Mammary pathogenic Escherichia coli (MPEC) is one of the most common pathogens associated with clinical mastitis. We analyzed isolates obtained from milk samples of cows with clinical mastitis, collected from 10 farms in Brazil, to verify molecular and phenotypic characteristics. A total of 192 (4.5%) mammary pathogenic E. coli isolates were obtained from 4,275 milk samples analyzed, but we tested 161. We assigned most of these isolates to E. coli phylogroups B1 (52.8%) and A (36.6%), although phylogroups B2, C, D, E, and unknown also occurred. All isolates were assessed for the presence of several genes encoding virulence factors, such as adhesins (sfaDE, papC, afaBC III, ecpA, fimH, papA, and iha), toxins (hlyA, cnf1, sat, vat, and cdt), siderophores (iroN, irp2, iucD, ireA, and sitA), an invasion protein (ibeA), and serum resistance proteins (traT, KpsMTII, and ompT), and isolates from phylogroups B1, B2, and E showed up to 8 genes. Two isolates harbored the locus of enterocyte effacement (escN+) and lack the bundle-forming pilus (bfpB-) operon, which corresponds to a molecular profile of a subgroup of diarrheagenic E. coli (aEPEC), thus being classified as hybrid MPEC/aEPEC isolates. These isolates displayed a localized adherence-like pattern of adherence in HeLa cells and were able to promote F-actin polymerization underneath adherent bacteria. Based on the pulsed-field gel electrophoresis analyses, considerable genetic variability was observed. A low index of antimicrobial resistance was observed and 2 extended-spectrum ß-lactamase-producing E. coli were identified, both harboring blaCTX-M15 gene, and were classified as ST10 and ST993 using multilocus sequence typing. A total of 148 (91.2%) isolates were weak biofilm producers or formed no biofilm. Because raw milk is still frequently consumed in Brazil, the occurrence of virulence factor-encoding genes from extraintestinal or diarrheagenic E. coli added to the presence of extended-spectrum ß-lactamase-producing isolates can turn this veterinary medicine problem into a public health concern.

Virulence-encoding genes related to extraintestinal pathogenic E. coli and multidrug resistant pattern of strains isolated from neonatal calves with different severity scores of umbilical infections.

Umbilical infections in calves comprise a major cause of neonatal mortality and have been related to a variety of microorganisms. E. coli is an opportunistic enteropathogen characterized by a diversity of virulence factors (VF). Nonetheless, the gene profiles that encode VF associated with umbilical infections in calves and their effect on the clinical severity remains unclear. In this scenario, microbial identification (with an emphasis on E. coli), was carried out among 150 neonatal calves (≤30 days of age) with umbilical infections, where the omphalopathies were clinically scored as mild, moderate, or severe. Also, a panel of 16 virulence-encoding genes related to extraintestinal pathogenic E. coli (ExPEC) were investigated, i.e., fimbriae/adhesins (sfa/focDEa, papA, papC, afaBC), toxins (hlyA, sat, cnf1, cdt), siderophores (iroN, irp2, iucD, ireA), invasins (ibeA), and serum resistance (ompT, traT, kpsMT II). Bacteria and yeasts isolates were identified using mass spectrometry. Bacteria, yeasts, and fungi were isolated in 94.7% (142/150) of neonatal calves sampled. E. coli was the agent most frequently isolated (59/150 = 39.3%), in pure culture (27/59 = 45.8%) and combined infections (32/59 = 54.2%), although a great variety (n = 83) of other species of microorganisms were identified. Clinical severity scores of 1, 2, and 3 were observed in 32.2% (19/59), 23.7% (14/59), and 44.1% (26/59) of E. coli infections, respectively. The ExPEC genes detected were related to serum resistance (traT, 42/59 = 72.2%; ompT, 35/59 = 59.3%, kpsMTII, 10/59 = 17%), invasins (ibeA, 11/59 = 18.6%), siderophores (iucD, 9/59 = 15.3%; iroN, 8/59 = 13.6%), and adhesins/fimbriae (papA, 8/59 = 13.6%; papC, 15/59 = 9.6%). The presence of each virulence gene was not associated with the case's clinical score. Among all isolates, 89.8% (53/59) showed in vitro resistance to sulfamethoxazole/trimethoprim and 59.3% to ampicillin (35/59), while 94.1% (55/59) revealed a multidrug resistant profile. Great complexity of bacteria, yeast, and fungi species was identified, reinforcing the umbilical infections of neonatal calves as a polymicrobial disorder. The high occurrence of E. coli (39.3%) highlights the role of this pathogen in the etiology of umbilical infections in calves. Furthermore, a panel of ExPEC genes was investigated for the first time among calves that were clinically scored for case severity. The high prevalence of traT and ompT indicates that these serum resistance-related genes could be used as biomarkers for further investigations of ExPEC isolates from umbilical infections. Our results contribute to the etiological investigation, clinical severity scoring, antimicrobial resistance pattern, and virulence-related to ExPEC genes involved in umbilical infections of neonatal calves.

Characterization of Uropathogenic Escherichia coli Reveals Hybrid Isolates of Uropathogenic and Diarrheagenic (UPEC/DEC) E. coli.

(1) Background: Pathogenic Escherichia coli are divided into two groups: diarrheagenic (DEC) and extraintestinal pathogenic (ExPEC) E. coli. ExPEC causing urinary tract infections (UTIs) are termed uropathogenic E. coli (UPEC) and are the most common cause of UTIs worldwide. (2) Methods: Here, we characterized 112 UPEC in terms of phylogroup, serotype, the presence of virulence factor-encoding genes, and antimicrobial resistance. (3) Results: The majority of the isolates were assigned into the phylogroup B2 (41.07%), and the serogroups O6 (12.5%) and O25 (8.9%) were the most frequent. Five hybrid UPEC (4.5%), with markers from two DEC pathotypes, i.e., atypical enteropathogenic (aEPEC) and enteroaggregative (EAEC) E. coli, were identified, and designated UPEC/aEPEC (one isolate) and UPEC/EAEC (four isolates), respectively. Three UPEC/EAEC harbored genes from the pap operon, and the UPEC/aEPEC carried ibeA. The highest resistance rates were observed for ampicillin (46.4%) and trimethoprim/sulfamethoxazole (34.8%), while 99.1% of the isolates were susceptible to nitrofurantoin and/or fosfomycin. Moreover, 9.8% of the isolates were identified as Extended Spectrum ß-Lactamase producers, including one hybrid UPEC/EAEC. (4) Conclusion: Our data reinforce that hybrid UPEC/DEC are circulating in the city of Botucatu, Brazil, as uropathogens. However, how and whether these combinations of genes influence their pathogenicity is a question that remains to be elucidated.

Identification of a hybrid atypical enteropathogenic and enteroaggregative Escherichia coli (aEPEC/EAEC) clone of serotype O3:H2 associated with a diarrheal outbreak in Brazil.

Enteropathogenic (EPEC) and enteroaggregative (EAEC) Escherichia coli are two of the major pathotypes of diarrheagenic E. coli causing disease worldwide. Here, we report a diarrheal outbreak caused by E. coli of serotype O3:H2, harboring virulence markers from EPEC (eae) and/or EAEC (aggR). This is likely the first E. coli diarrheal outbreak caused by a hybrid atypical-EPEC/EAEC clone reported in Brazil.

Short communication: Investigation of extra-intestinal pathogenic Escherichia coli virulence genes, bacterial motility, and multidrug resistance pattern of strains isolated from dairy cows with different severity scores of clinical mastitis.

Escherichia coli is a major pathogen involved in the etiology of environmentally derived bovine mastitis and is characterized by a variety of virulence factors (VF). Mammary infections with E. coli have shown a wide range of clinical signs, causing changes in milk (score 1, or mild), abnormal appearance of milk and udder inflammation (score 2, or moderate), and abnormalities in milk, udder inflammation, and systemic signs of illness (score 3, or severe). Nevertheless, to date, the profile of the genes related to the virulence of the pathogen in mammary infections and the severity scores of cases have not been thoroughly elucidated. Therefore, a panel of 18 virulence-encoding genes associated with extra-enteric pathogenicity of E. coli (ExPEC) were investigated in addition to in vitro swimming and swarming motility profiles and antimicrobial susceptibility/resistance patterns among 114 E. coli strains isolated from cows with clinical mastitis and different severity scores. Of 114 clinical cases, 39.5, 54.4, and 6.1% were mild, moderate, and severe, respectively. The main genes related to VF harbored by isolates were adhesins (fimH 100%; ecpA 64.0%, fimA 31.6%), serum resistance (traT 81.6%; ompT 35.1%), siderophores (irp2 9.6%), and hemolysin (hlyA 7%). Among the isolates studied, 99.1% showed in vitro resistance to bacitracin and cloxacillin, and 98.2% to lincosamin. Of the total isolates, 98.2% were considered multidrug resistant based on the multiple antimicrobial resistance index. No significant difference was observed between mean swimming (13.8 mm) and swarming (13.5 mm) motility, as well as severity scores of clinical mastitis and the ExPEC genes studied. The isolation of strains resistant to various antimicrobials, even though tested only in vitro, highlights the importance of rational use of antimicrobials for mastitis treatment. The high prevalence of the genes related to serum resistance (traT and ompT) and adhesion (ecpA) of the pathogen, in addition to main associations between the genes fimH, ecpA, and traT among cows with severity scores of 1 (15%) and 2 (22.6%), indicates that the genes traT, ecpA, and ompT could be further studied as biomarkers of ExPEC for clinical intramammary infections. In addition, the ExPEC genes ompT (protectin), ibe10 (invasin), and ecpA (adhesin) were investigated for the first time among cows with mastitis, where scores of clinical severity were assessed. Results of this study contribute to the characterization of virulence mechanisms and antimicrobial resistance profile of ExPEC variants that affect dairy cows with different scores of clinical mastitis.