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Introduction: Veterinary clinical microbiology laboratories play a key role in antimicrobial stewardship, surveillance of antimicrobial resistance and prevention of healthcare associated-infections. However, there is a shortage of international harmonized guidelines covering all steps of veterinary bacterial culture from sample receipt to reporting., Methods: In order to gain insights, the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT) designed an online survey focused on the practices and interpretive criteria used for bacterial culture and identification (C&ID), and antimicrobial susceptibility testing (AST) of animal bacterial pathogens., Results: A total of 241 microbiology laboratories in 34 European countries completed the survey, representing a mixture of academic (37.6%), governmental (27.4%), and private (26.5%) laboratories. The C&ID turnaround varied from 1 to 2 days (77.8%) to 3-5 days (20%), and 6- 8 days (1.6%), with similar timeframes for AST. Individual biochemical tests and analytical profile index (API) biochemical test kits or similar were the most frequent tools used for bacterial identification (77% and 56.2%, respectively), followed by PCR (46.6%) and MALDI-TOF MS (43.3%). For AST, Kirby-Bauer disk diffusion (DD) and minimum inhibitory concentration (MIC) determination were conducted by 43.8% and 32.6% of laboratories, respectively, with a combination of EUCAST and CLSI clinical breakpoints (CBPs) preferred for interpretation of the DD (41.2%) and MIC (47.6%) results. In the absence of specific CBPs, laboratories used human CBPs (53.3%) or veterinary CBPs representing another body site, organism or animal species (51.5%). Importantly, most laboratories (47.9%) only report the qualitative interpretation of the result (S, R, and I). As regards testing for AMR mechanisms, 48.5% and 46.7% of laboratories routinely screened isolates for methicillin resistance and ESBL production, respectively. Notably, selective reporting of AST results (i.e. excluding highest priority critically important antimicrobials from AST reports) was adopted by 39.5% of laboratories despite a similar proportion not taking any approach (37.6%) to guide clinicians towards narrower-spectrum or first-line antibiotics., Discussion: In conclusion, we identified a broad variety of methodologies and interpretative criteria used for C&ID and AST in European veterinary microbiological diagnostic laboratories. The observed gaps in veterinary microbiology practices emphasize a need to improve and harmonize professional training, innovation, bacterial culture methods and interpretation, AMR surveillance and reporting strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Koritnik, Cvetkovikj, Zendri, Blum, Chaintoutis, Kopp, Hare, Štritof, Kittl, Gonçalves, Zdovc, Paulshus, Laconi, Singleton, Allerton, Broens, Damborg and Timofte.)

The increasing prevalence of antibiotic-resistant bacteria is an emerging threat to global health. The analysis of antibiotic-resistant enterobacteria in wastewater can indicate the prevalence and spread of certain clonal groups of multiresistant bacteria. In a previous study of Escherichia coli that were isolated from a pump station in Norway over 15 months, we found a recurring E. coli clone that was resistant to trimethoprim, ampicillin, and tetracycline in 201 of 3,123 analyzed isolates (6.1%). 11 representative isolates were subjected to whole-genome sequencing and were found to belong to the MLST ST2797 E. coli clone with plasmids carrying resistance genes, including bla TEM-1B , sul2 , dfrA7 A phenotypic comparison of the ST2797 isolates with the uropathogenic ST131 and ST648 that were repeatedly identified in the same wastewater samples revealed that the ST2797 isolates exhibited a comparable capacity for temporal survival in wastewater, greater biofilm formation, and similar potential for the colonization of mammalian epithelial cells. ST2797 has been isolated from humans and has been found to carry extended spectrum β-lactamase (ESBL) genes in other studies, suggesting that this clonal type is an emerging ESBL E. coli. Collectively, these findings show that ST2797 was more ubiquitous in the studied wastewater than were the infamous ST131 and ST648 and that ST2797 may have similar abilities to survive in the environment and cause infections in humans. tetB. The incidence of drug-resistant bacteria found in the environment is increasing together with the levels of antibiotic-resistant bacteria that cause infections. The COVID-19 pandemic has shed new light on the importance of monitoring emerging threats and finding early warning systems. Therefore, to mitigate the antimicrobial resistance burden, the monitoring and early identification of antibiotic-resistant bacteria in hot spots, such as wastewater treatment plants, are required to combat the occurrence and spread of antibiotic-resistant bacteria. Here, we applied a PhenePlate system as a phenotypic screening method for genomic surveillance and discovered a dominant and persistent E. coli clone ST2797 with a multidrug resistance pattern and equivalent phenotypic characteristics to those of the major pandemic lineages, namely, ST131 and ST648, which frequently carry ESBL genes. This study highlights the continuous surveillance and report of multidrug resistant bacteria with the potential to spread in One Health settings.IMPORTANCE The incidence of drug-resistant bacteria found in the environment is increasing together with the levels of antibiotic-resistant bacteria that cause infections. The COVID-19 pandemic has shed new light on the importance of monitoring emerging threats and finding early warning systems. Therefore, to mitigate the antimicrobial resistance burden, the monitoring and early identification of antibiotic-resistant bacteria in hot spots, such as wastewater treatment plants, are required to combat the occurrence and spread of antibiotic-resistant bacteria. Here, we applied a PhenePlate system as a phenotypic screening method for genomic surveillance and discovered a dominant and persistent E. coli clone ST2797 with a multidrug resistance pattern and equivalent phenotypic characteristics to those of the major pandemic lineages, namely, ST131 and ST648, which frequently carry ESBL genes. This study highlights the continuous surveillance and report of multidrug resistant bacteria with the potential to spread in One Health settings., Competing Interests: The authors declare no conflict of interest.

A comprehensive monitoring of a broad set of antibiotics in the final effluent of wastewater treatment plants (WWTPs) of 7 European countries (Portugal, Spain, Ireland, Cyprus, Germany, Finland, and Norway) was carried out in two consecutive years (2015 and 2016). This is the first study of this kind performed at an international level. Within the 53 antibiotics monitored 17 were detected at least once in the final effluent of the WWTPs, i.e.: ciprofloxacin, ofloxacin, enrofloxacin, orbifloxacin, azithromycin, clarithromycin, sulfapyridine, sulfamethoxazole, trimethoprim, nalidixic acid, pipemidic acid, oxolinic acid, cefalexin, clindamycin, metronidazole, ampicillin, and tetracycline. The countries exhibiting the highest effluent average concentrations of antibiotics were Ireland and the southern countries Portugal and Spain, whereas the northern countries (Norway, Finland and Germany) and Cyprus exhibited lower total concentration. The antibiotic occurrence data in the final effluents were used for the assessment of their impact on the aquatic environment. Both, environmental predicted no effect concentration (PNEC-ENVs) and the PNECs based on minimal inhibitory concentrations (PNEC-MICs) were considered for the evaluation of the impact on microbial communities in aquatic systems and on the evolution of antibiotic resistance, respectively. Based on this analysis, three compounds, ciprofloxacin, azithromycin and cefalexin are proposed as markers of antibiotic pollution, as they could occasionally pose a risk to the environment. Integrated studies like this are crucial to map the impact of antibiotic pollution and to provide the basis for designing water quality and environmental risk in regular water monitoring programs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bacterial diversity and antimicrobial resistance patterns among the indicator organism Escherichia coli were monitored in wastewater samples collected over one year from a hospital (HW), a community (CW) and the receiving urban (UW) wastewater treatment plant (WWTP). We compared levels of antibiotic resistance in the different types of wastewater, and identified whether resistant strains were endemic in the wastewater system. If so, implementation of local treatment at certain resistance hotspots (e.g. hospital outlets) could be used to decrease the amount of resistant bacteria in the wastewater. E. coli from HW (n = 2644), CW (n = 2525) and UW (n = 2693) were analyzed by biochemical phenotyping (PhenePlate System) and antimicrobial susceptibility testing to nine antibiotics (AREB System). The phenotypic diversities of the total E. coli populations were similar for all three sites (Simpson's Diversity index, Di = 0.973), however for individual samples, HW showed low diversities (Median Di = 0.800) and the E. coli flora was often dominated by strains that may have originated from the fecal flora of single individuals. The diversities in CW samples was higher (Median Di = 0.936), and UW samples showed similar diversities as the whole collection of isolates (Median Di = 0.971). Resistance to at least one of the nine antibiotics was observed in 45% of the HW isolates, 44% of CW isolates, and 33% of UW isolates. Resistance to gentamicin and chloramphenicol was uncommon (3.2 and 5.3%, respectively), whereas resistance to tetracycline and ampicillin was most common (24% and 31%, respectively). Extended-spectrum beta-lactamase-producing E. coli (ESBL-EC) were more common in HW (11.5%) and in CW (6.9%) compared to UW (3.7%). A high diversity (Di = 0.974) was observed among ESBL-EC isolates from UW (n = 99), indicating absence of any clonal structure among these isolates. Common PhP types of ESBL-EC often dominated in each HW sample, but were not identified across different samples, whereas ESBL-EC in CW showed low diversity (Di = 0.857) and were dominated by a specific PhP type that was found across almost all CW samples. The antibiotic resistance rates were highest in hospital wastewater, but surprisingly they were also high in the studied community wastewater, compared to the urban wastewater. The relative contribution of HW seemed low in terms of dissemination of antibiotic resistant bacteria to the WWTP., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Antibiotic resistance in bacteria is an emerging problem globally. Resistant bacteria are found in human and animal microbiota, as well as in the environment. Wastewater receives bacteria from all these sources and thus can provide a measurement of abundance and diversity of antibiotic-resistant bacteria circulating in communities. In this study, water samples were collected from a wastewater pump station in a Norwegian suburban community over a period of 15 months. A total of 45 daily samples were cultured and analyzed for the presence of Escherichia coli Eighty E. coli -like colonies were collected from each daily sample and then phenotyped and analyzed for antibiotic resistance using the PhenePlate-AREB system. During the sampling period, two unique E. coli phenotypes with resistance to cefotaxime and cefpodoxime indicating carriage of extended-spectrum β-lactamases (ESBL) were observed repeatedly. Whole-genome sequencing of 15 representative isolates from the two phenotypes identified these as two distinct clones belonging to the two globally spread E. coli multilocus sequence types (STs) ST131 and ST648 and carrying bla CTX-M-15 The number of ESBL-positive E. coli strains in the community wastewater pump station was 314 of 3,123 (10%) analyzed E. coli strains. Of the ESBL-positive isolates, 37% belonged to ST648, and 7% belonged to ST131. Repeated findings of CTX-M-15-positive ST648 and ST131 over time indicate that these STs are resident in the analyzed wastewater systems and/or circulate abundantly in the community., (Copyright © 2019 Paulshus et al.)

Aliivibrio salmonicida is the causative agent of cold-water vibriosis, a septicemia of farmed salmonid fish. The mechanisms of disease are not well described, and few virulence factors have been identified. However, a requirement for motility in the pathogenesis has been reported. Al. salmonicida is motile by the means of lophotrichous polar flagella, consisting of multiple flagellin subunits that are expressed simultaneously. Here we show that flagellin subunit FlaA, but not FlaD, is of major importance for motility in Al. salmonicida. Deletion of flaA resulted in 62% reduction in motility, as well as a reduction in the fraction of flagellated cells and number of flagella per cell. Similarly, deletion of the gene encoding motor protein motA gave rise to an aflagellate phenotype and cessation of motility. Surprisingly, we found that Al. salmonicida does not require motility for invasion of Atlantic salmon. Nevertheless, in-frame deletion mutants defective of motA and flaA were less virulent in Atlantic salmon challenged by immersion, whereas an effect on virulence after i.p. challenge was only seen for the latter. Our results indicate a complex requirement for motility and/or flagellation in the pathogenesis of cold-water vibriosis, but the mechanisms involved remain unknown. We hypothesize that the differences in virulence observed after immersion and i.p. challenge are related to the immune response of the host., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)