Marisa Haenni

Background/Objectives: The emergence of plasmid-mediated mcr genes has enabled horizontal dissemination of resistance to colistin, a last-resort antibiotic against multidrug-resistant Enterobacterales. In Tunisia, genomic data on mcr-positive Escherichia coli are still limited. This study reports the genomic characterization of human clinical mcr-positive E. coli isolates from the Military Hospital of Tunis. Methods: Between August 2023 and March 2025, seven E. coli isolates with low-level colistin-resistance (MIC = 4–8 µg/mL) were collected from six patients. They were characterized by antibiotic susceptibility testing and WGS to determine resistome, MLST, genetic relatedness, and plasmid content. Results: The E. coli isolates belonged to diverse sequence types (STs), except for two isolates collected from the same patient 2.5 months apart, which were highly related. Overall, this pattern is consistent with a polyclonal spread. The mcr-1.1 gene was located on IncI2 (n = 5) or IncX4 (n = 2) plasmids, which exhibited high similarity both among themselves and in comparison with plasmids previously reported in human and livestock isolates. Most isolates were multidrug-resistant, harboring acquired resistance genes to multiple antibiotic classes, and chromosomal mutations conferring fluoroquinolone resistance. Three isolates additionally carried chromosomal insertions of the bla_CTX-M-55 gene. Resistance to cefiderocol was observed in one isolate and was associated with CirA and Fiu truncation. Conclusions: These findings highlight ongoing dissemination of mcr-1.1-positive E. coli isolates in Tunisia, primarily driven by plasmid transfer. Continuous genomic surveillance and One Health-oriented antibiotic stewardship are essential to limit the spread of colistin-resistance and the emergence of resistance to newer agents such as cefiderocol.

Antimicrobial resistance (AMR) is an important One Health issue affecting the three human, animal, and environmental sectors. The occurrence of Escherichia coli resistant to extended-spectrum cephalosporins (ESC-R) is considered a relevant marker for the estimation of the AMR burden. In the dairy industry, veal calves present the highest proportions of ESC-R compared to adults. Since little is known in France about the AMR dynamics from birth to the fattening farm, our observational study determined the proportion of ESC-R E. coli using selective media, and characterized by whole-genome sequencing. Bacterial isolates identified at the farm level and at the sorting center, before being sent to the fattening farms. The proportion of ESC-R-positive calves coming from farms in which waste milk was given to calves (30.3%) was higher than in the ones where it was discarded (13.1%). Overall, 12.5% of the veal calves were ESC-R carriers in their farm of origin, and 6.2% of the additional animals became carriers during their transport to the sorting center. Over the 176 ESC-R E. coli isolates sequenced, 55.1% ( n = 97) presented an extended-spectrum beta-lactamase phenotype conferred by a bla _CTX-M gene, while 44.9% ( n = 79) presented an AmpC phenotype due to chromosomal mutations. A few specific clones (ST8095 and ST117) were identified as responsible for AMR dissemination across farms. Our results showed that AMR in dairy calves results from cumulative effects of all risk factors along the whole production chain, including feeding calves with antibiotic residues-containing waste milk, animal grouping, and insufficient biosecurity measures in dairy farms. IMPORTANCE: Despite the implementation of national action plans, the presence of Escherichia coli resistant to extended-spectrum cephalosporins (ESC-R), mainly due to the presence of extended-spectrum beta-lactamases or AmpCs, is still high in many veal-producing countries. In France, previous studies reported antimicrobial resistance (AMR) data during the fattening period and at the slaughterhouse, but data are still scarce on the AMR status of veal calves on their farm of origin. This study aimed at determining the proportion of ESC-R E. coli and characterizing bacterial isolates identified at the farm level and at the sorting center, just before being sent to the fattening farms. This is of utmost importance since mathematical models showed that minimizing AMR prevalence upon arrival in the fattening farm is an important factor to mitigate ESC-R carriage during the fattening process. In addition, we are providing important WGS data from the veal calf sector, which can be compared to already-existing data.

In streptococci, the acquisition of high-level gentamicin resistance (HLGR) abolishes the synergy with beta-lactams, constituting a medical concern for humans and animals. This synergy between gentamicin and beta-lactams is useful to treat severe infections like endocarditis, among others. HLGR has been characterized mostly in roup B Streptococci mediated by the bi-functional gene aac (6)- aph (2). HLGR– Streptococcus dysgalactiae subsp. equisimilis ( SDSE ) ( n =27) were isolated from the uterus of mares during a routine screening before in vitro fecundation. One HLGR– SDSE was isolated from a lymph node of a mare. The mares resided in six different studs located in four departments of France. HLGR –SDSE belonged to three sequence types (STs): ST196 ( n =26), ST223 ( n =1) and a single locus variant (SLV) of ST200 ( n =1). For the first time, HLGR was found mediated by the two genes aph (2″)- Ib and aac (6’)- Iam located on a class 5 integrative mobilizable element (IME), here named HLGR_IME. In HLGR– SDSE assigned to ST196 and ST223, the HLGR_IME was inserted in a Tn 5252 -type transposon. In the isolate assigned to an ST200-SLV, the HLGR_IME was inserted in an ICE Er0106 -type element. The Tn 5252 -type transposon was able to transfer in vitro from HLGR –SDSE to an erythromycin-resistant Streptococcus agalactiae strain. The transferability of HLGR from SDSE to human pathogens, such as S. agalactiae , is a major concern for health. The phylogenetic analysis displayed that SDSE from horses constituted a distinct phylogenetic clade from SDSE isolated from humans or other animals. Furthermore, we demonstrated that HLGR– SDSE belonging to ST196 were able to disseminate among mares within a single stud during 2 years. This clone has also been reported in the USA. Hygiene measures could prevent further dissemination of HLGR streptococci in veterinary settings, preserving horses’ health, and the transfer of HLGR to pathogens important in veterinary and human settings.

Background and objectives: Neomycin is widely used in livestock to control colibacillosis. Resistance among clinical Escherichia coli is increasing, likely driven by the use of this aminoglycoside following the European restrictions on zinc oxide and colistin. The aim of this study was to investigate the genetic epidemiology of neomycin-resistant E. coli across six animal species and five European countries. Methods: A total of 750 neomycin-resistant E. coli isolates were included, combining retrospectively identified genomes with prospectively isolated and sequenced strains from multiple animal species. Genomic analyses, based on short reads (n = 529), long reads (n = 177), or hybrid assemblies (n = 44), included phylogenetic comparison, and identification of neomycin resistance genes and their genetic contexts. Results: Neomycin resistance was mediated by aph(3′)-Ia in 98.2% of the isolates, which were distributed across 28 clonal complexes, predominantly CC10, which was frequently detected in all countries and hosts. aph(3′)-Ia was primarily carried by conjugative plasmids of diverse replicon types (70.3%) and embedded in two transposons (Tn903 and Tn4352) and three newly identified genetic elements, while a chromosomally integrated element accounted for the remaining 29.7%. Plasmids carrying aph(3′)-Ia frequently co-harboured tetracycline and trimethoprim-sulfamethoxazole resistance genes, with IncX1 plasmids additionally containing chloramphenicol and quinolone resistance determinants. Phylogenetic analysis revealed dissemination of plasmid-borne elements across multiple lineages, while the chromosomal element was mostly restricted to ST117, ST88, and ST189. Conclusions: The spread of neomycin resistance in E. coli from European livestock is primarily driven by the mobility of aph(3′)-Ia embedded in diverse genetic contexts and plasmid backbones, promoting co-transfer of other resistance determinants.

The increase in the infection caused by Acinetobacter baumannii is sustained by the selection of distinct epidemic clonal lineages, which are frequently resistant to a broad range of antimicrobials and possess virulence traits responsible for their persistence in the contaminated environment and spread among patients. The present study aimed to perform an integrated genomic and phenotypic analysis to assess the virulence features of ST25 isolates. A. baumannii isolates assigned to the ST25 epidemic clonal lineage shared high genomic similarity and clustered in four clades (I, II, III, and IV), with clade IV further subdivided into CIVa, CIVb, CIVc, and CIVd. Capsular locus (KL) KL14 was the predominant KL type (47%). Accessory genome analysis showed the presence of tartrate metabolism genes only in CII genomes. CIVb and CIVd ST25 A. baumannii isolates showed higher ability to infect Galleria mellonella larvae than CI, CII, CIII, and CIVc isolates. Hydrogen peroxide resistance was higher in CI, CII, CIVb, and CIVd isolates compared with CIII and CIVc isolates. In desiccation survival tests, CIII, CIVb, and CIVd isolates exhibited prolonged survival. In addition, CI, CII, CIII, CIVb, and CIVd isolates showed higher serum resistance than CIVc isolates. Also, KL14 type and lipooligosaccharide outer core locus (OCL) OCL6 type isolates were significantly more resistant to oxidative stress, to desiccation, and possessed a high ability to kill G. mellonella larvae. A positive and significant correlation was found between AdeB and AdeJ efflux pump expression and hydrogen peroxide resistance. IMPORTANCE: In this study, we characterized the genotypic and phenotypic features of A. baumannii strains assigned to the ST25 epidemic clonal lineage, which were isolated from humans, animals, and the environment. We found that ST25 A. baumannii isolates, irrespective of their antimicrobial resistance, showed peculiar virulence features among clades, isolates assigned to clade IVb and IVd showing the highest virulence and elevated resistance to serum and desiccation. Also, a positive significant correlation was found between the presence of KL14 and outer core locus 6 genotypes and resistance to oxidative stress, resistance to desiccation, and the ability to kill G. mellonella larvae. Phenotypic differences reflected clade identity rather than isolate origin, suggesting that specific virulence traits contribute to the environmental persistence and pathogenic potential of A. baumannii ST25 isolates.

Amoxicillin-clavulanic acid (AMC) is an important antibiotic in human and veterinary medicine. However, it can select antibiotic-resistant bacteria in the gut microbiota and alter its composition. In calves, AMC can be used to treat complicated infections. The impact of AMC on the calves’ gut is unknown; thus, we analyzed the effect of a five-day AMC treatment in veal calves suffering from omphalitis. AMC-treated calves ( n = 15) and a control untreated group ( n = 15) were enrolled from two farms. Stools were collected before AMC administration (D0), one day (D6), one month (D35), and two months (D55) after AMC withdrawal. The effect of AMC treatment on gut microbiota composition and resistance gene selection was analyzed by full-length 16S rRNA operon sequencing and qPCR, respectively. Selection of Extended-Spectrum Cephalosporin-Resistant (ESC-R) Escherichia coli in calf’s fecal samples and dissemination in the farms’ environment were analyzed by cultivation and genome sequencing of isolates. After AMC treatment, alpha-diversity decreased in AMC-treated calves along with a decrease of the relative abundance of bacterial families beneficial for host health (Lachnospiraceae) and an increase of Pseudomonadota, grouping opportunistic pathogens. ESC-R E. coli carriage dynamics were different between calves sampled from each farm and seemed independent from AMC treatment. Besides, calves received other antibiotics that could have an impact on resistance selection. Certain clones of ESC-R E. coli demonstrated a widespread dissemination in the farm, both in calves hosted in distant zones and in their environment. To decrease the antibiotic resistance burden, it is essential to use antibiotics judiciously, alongside improving hygienic practices in farms. IMPORTANCE: Antibiotic therapies can select resistant bacteria in the gut of treated hosts and deplete bacteria that are beneficial to the host health. Antibiotic-resistant bacteria selected in the gut of food-producing animals, like veal calves, are excreted and can then disseminate among animals, to the environment (through manure or water contamination) and to farmers who may further disseminate these organisms to other people in contact. Antibiotic resistance genes can disseminate among clones present in the gut of both animal and human hosts by horizontal gene transfer. Studying the impact of antibiotic therapies on the gut microbiota has One Health relevance. Thus, we aimed to (i) analyze the impact of AMC treatment on the selection of resistant bacteria in the calf gut and its composition and (ii) analyze the dissemination resistance in farms in order to advise on potential strategies to counteract further spread of these microorganisms.