Enteroaggregative Escherichia coli: Frequent, yet underdiagnosed pathotype among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic.

Enteroaggregative Escherichia coli (EAEC) strains including those of serogroup O111 are important causes of diarrhea in children. In the Czech Republic, no information is available on the etiological role of EAEC in pediatric diarrhea due to the lack of their targeted surveillance. To fill this gap, we determined the proportion of EAEC among E. coli O111 isolates from children with gastrointestinal disorders ≤ 2 years of age submitted to the National Reference Laboratory for E. coli and Shigella during 2013-2022. EAEC accounted for 177 of 384 (46.1 %) E. coli O111 isolates, being the second most frequent E. coli O111 pathotype. Most of them (75.7 %) were typical EAEC that carried aggR, usually with aaiC and aatA marker genes; the remaining 24.3 % were atypical EAEC that lacked aggR but carried aaiC and/or aatA. Whole genome sequencing of 11 typical and two atypical EAEC O111 strains demonstrated differences in serotypes, sequence types (ST), virulence gene profiles, and the core genomes between these two groups. Typical EAEC O111:H21/ST40 strains resembled by their virulence profiles including the presence of the aggregative adherence fimbriae V (AAF/V)-encoding cluster to such strains from other countries and clustered with them in the core genome multilocus sequence typing (cgMLST). Atypical EAEC O111:H12/ST10 strains lacked virulence genes of typical EAEC and differed from them in cgMLST. All tested EAEC O111 strains displayed stacked-brick aggregative adherence to human intestinal epithelial cells. The AAF/V-encoding cluster was located on a plasmid of 95,749 bp or 93,286 bp (pAAO111) which also carried aggR, aap, aar, sepA, and aat cluster. EAEC O111 strains were resistant to antibiotics, in particular to aminopenicillins and cephalosporins; 88.3 % produced AmpC ß-lactamase, and 4.1 % extended spectrum ß-lactamase. We conclude that EAEC are frequent among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic. To reliably assess the etiological role of EAEC in pediatric diarrhea, a serotype-independent, PCR-based pathotype surveillance system needs to be implemented in the future.

Translocation of outer membrane vesicles from enterohemorrhagic Escherichia coli O157 across the intestinal epithelial barrier.

Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS.

In Vivo Secretion of ß-Lactamase-Carrying Outer Membrane Vesicles as a Mechanism of ß-Lactam Therapy Failure.

Outer membrane vesicles carrying ß-lactamase (ßLOMVs) protect bacteria against ß-lactam antibiotics under experimental conditions, but their protective role during a patient's treatment leading to the therapy failure is unknown. We investigated the role of ßLOMVs in amoxicillin therapy failure in a patient with group A Streptococcus pyogenes (GAS) pharyngotonsillitis. The patient's throat culture was examined by standard microbiological procedures. Bacterial vesicles were analyzed for ß-lactamase by immunoblot and the nitrocefin assay, and in vivo secretion of ßLOMVs was detected by electron microscopy. These analyses demonstrated that the patient's throat culture grew, besides amoxicillin-susceptible GAS, an amoxicillin-resistant nontypeable Haemophilus influenzae (NTHi), which secreted ßLOMVs. Secretion and ß-lactamase activity of NTHi ßLOMVs were induced by amoxicillin concentrations reached in the tonsils during therapy. The presence of NTHi ßLOMVs significantly increased the minimal inhibitory concentration of amoxicillin for GAS and thereby protected GAS against bactericidal concentrations of amoxicillin. NTHi ßLOMVs were identified in the patient's pharyngotonsillar swabs and saliva, demonstrating their secretion in vivo at the site of infection. We conclude that the pathogen protection via ßLOMVs secreted by the flora colonizing the infection site represents a yet underestimated mechanism of ß-lactam therapy failure that warrants attention in clinical studies.

A cross-border outbreak of Salmonella Bareilly cases confirmed by whole genome sequencing, Czech Republic and Slovakia, 2017 to 2018.

In August 2017, an increased incidence of Salmonella Bareilly was detected in the Czech Republic. An investigation was conducted with Slovakia to confirm the outbreak and identify the source. Probable outbreak cases were defined as cases with laboratory-confirmed S. Bareilly reported in either of the national surveillance systems, and/or the Czech and Slovak National Reference Laboratory databases from July 2017. Confirmed cases had the pulsed-field gel electrophoresis (PFGE) outbreak pulsotype or up to 5 alleles difference from outbreak cluster members by core genome multilocus sequence typing (cgMLST). PFGE and whole genome sequencing were used for isolate comparison. The same trawling questionnaire was used in both countries. By the end of October 2018, 325 cases were identified. Among 88 human S. Bareilly isolates analysed by PFGE, 82 (93%) shared an identical pulsotype; cgMLST of 17 S. Bareilly human isolates showed 1-2 allele difference. The trawling questionnaire excluded consumption of unusual or imported foods. In September 2018, an isolate closely related to the outbreak isolates was identified in a powdered egg product. A spray dryer was recognised as the contamination source and the production plant was closed. Using molecular typing methods, we detected a diffuse cross-border outbreak caused by S. Bareilly.

Dissemination of the blaCTX-M-15 gene among Enterobacteriaceae via outer membrane vesicles.

BACKGROUND: Bacterial outer membrane vesicles (OMVs) are an emerging source of antibiotic resistance transfer but their role in the spread of the blaCTX-M-15 gene encoding the most frequent CTX-M ESBL in Enterobacteriaceae is unknown. OBJECTIVES: To determine the presence of blaCTX-M-15 and other antibiotic resistance genes in OMVs of the CTX-M-15-producing MDR Escherichia coli O104:H4 outbreak strain and the ability of these OMVs to spread these genes among Enterobacteriaceae under different conditions. METHODS: OMV-borne antibiotic resistance genes were detected by PCR; OMV-mediated transfer of blaCTX-M-15 and the associated blaTEM-1 was quantified under laboratory conditions, simulated intraintestinal conditions and under ciprofloxacin stress; resistance to antibiotics and the ESBL phenotype were determined by the CLSI disc diffusion methods and the presence of pESBL by plasmid profiling and Southern blot hybridization. RESULTS: E. coli O104:H4 OMVs carried blaCTX-M-15 and blaTEM-1 located on the pESBL plasmid, but not chromosomal antibiotic resistance genes. The OMVs transferred blaCTX-M-15, blaTEM-1 and the associated pESBL into Enterobacteriaceae of different species. The frequencies of the OMV-mediated transfer were significantly increased under simulated intraintestinal conditions and under ciprofloxacin stress when compared with laboratory conditions. The 'vesiculants' (i.e. recipients that received the blaCTX-M-15- and blaTEM-1-harbouring pESBL via OMVs) acquired resistance to cefotaxime, ceftazidime and cefpodoxime and expressed the ESBL phenotype. They were able to further spread pESBL and the blaCTX-M-15 and blaTEM-1 genes via OMVs. CONCLUSIONS: OMVs are efficient vehicles for dissemination of the blaCTX-M-15 gene among Enterobacteriaceae and may contribute to blaCTX-M-15 transfer in the human intestine.

Determination of factors associated with natural soil suppressivity to potato common scab.

Common scab of potatoes is a disease, which is difficult to manage due to complex interactions of the pathogenic bacteria (Streptomyces spp.) with soil, microbial community and potato plants. In Bohemian-Moravian Highlands in the Czech Republic two sites (Vyklantice and Zdirec) were selected for a study of common scab disease suppressivity. At both sites, a field with low disease severity occurs next to one with high severity and the situation was regularly observed over four decades although all four fields undergo a crop rotation. In the four fields, quantities of bacteria, actinobacteria and the gene txtB from the biosynthetic gene cluster of thaxtomin, the main pathogenicity factor of common scab, were analyzed by real-time PCR. Microbial community structure was compared by terminal fragment length polymorphism analysis. Soil and potato periderm were characterized by contents of carbon, nitrogen, phosphorus, sulphur, calcium, magnesium, and iron. Quality of organic matter was assessed by high performance liquid chromatography of soil extracts. The study demonstrated that the suppressive character of the fields is locally specific. At Zdirec, the suppressivity was associated with low txtB gene copies in bulk soil, while at Vyklantice site it was associated with low txtB gene copies in the tuberosphere. The differences were discussed with respect to the effect of abiotic conditions at Zdirec and interaction between potato plant and soil microbial community at Vyklantice. Soil pH, Ca soil content or cation concentrations, although different were not in the range to predict the disease severity. Low severity of common scab was associated with low content of soil C, N, C/N, Ca and Fe suggesting that oligotrophic conditions may be favorable to common scab suppression.