Beatriz Oporto

Food‐producing animals act as reservoirs of antimicrobial‐resistant bacteria, which may serve as sources of infection in humans. A selection of ESBL/AmpC‐producing E. coli isolates recovered from healthy ( n = 37) and diseased animals ( n = 26) were subjected to long‐read whole‐genome sequencing to characterize their genomic diversity, phylogeny, resistome, mobilome, and the genetic environment of antimicrobial resistance genes (ARGs). Isolates showed high genetic diversity (36 STs, seven phylogroups, 42 serotypes, 24 fimH types) and a diverse resistome, with CTX‐M enzymes as the most prevalent ESBL type and bla _CTX‐M‐14 and bla _CTX‐M‐65 predominating. Most ARGs were plasmid‐encoded (71.9%), with IncHI2 plasmids harboring the highest number of ARGs (8–20) and carrying complex multidrug resistance islands interspersed with integrons, transposons, and insertion sequences. All 15 IncHI2 plasmids were found exclusively in bovine isolates of multiple phylogroups (5) and STs (13), and most (13/15) carried bla _CTX‐M‐65 . This finding suggests a dissemination of this ESBL gene through a shared plasmid rather than a single dominant clone. This study reveals an emerging circulation of bla _CTX‐M‐65 in bovine E. coli in Spain and identifies IncHI2 plasmids as major vehicles driving its spread. The presence of these plasmids in genetically diverse and potentially zoonotic linages reinforces their relevance from a One Health and food‐chain perspective.

Enterococcus faecalis ( Efs ) and Enterococcus faecium ( Efm ) are major causes of multiresistant healthcare-associated or nosocomial infections. Efm has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to Enterococcus lactis ( Elc ). However, identification techniques do not routinely differentiate Elc from Efm . As part of a longitudinal study to investigate the antimicrobial resistance of Enterococcus in dairy cattle, isolates initially identified as Efm were confirmed as Elc after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An Efm -specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 Efs , 23 Efm , and 59 Elc . Resistance, determined by broth microdilution, was more frequent in Efs than in Efm and Elc but all isolates were susceptible to ampicillin, daptomycin, teicoplanin, tigecycline, and vancomycin. Genome sequencing analysis of 32 isolates identified 23 antimicrobial resistance genes (ARGs, mostly plasmid-located) and 2 single nucleotide polymorphisms associated with resistance to 10 antimicrobial classes, showing high concordance with phenotypic resistance. Notably, linezolid resistance in Efm was encoded by the optrA gene, located in plasmids downstream of the fexA gene. Although most Elc lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that Elc is more prevalent than Efm in dairy cattle but carries fewer ARGs and virulence genes. However, Elc can carry multi-drug-resistant plasmids like those harbored by Efm and could act as a donor of ARGs for other pathogenic enterococcal species. IMPORTANCE: Enterococcus species identification is crucial due to differences in pathogenicity and antibiotic resistance profiles. The failure of traditional methods or whole-genome sequencing-based taxonomic classifiers to distinguish Enterococcus lactis ( Elc ) from Enterococcus faecium ( Efm ) results in a biased interpretation of Efm epidemiology. The Efm species-specific real-time PCR assay developed here will help to properly identify Efm (only the formerly known clade A) in future studies. Here, we showed that Elc is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than Enterococcus faecalis ( Efs ) and Efm , it can carry multi-drug-resistant (MDR) plasmids and could act as a donor of resistance genes for other pathogenic enterococcal species. Although all isolates ( Efs , Efm , and Elc ) were susceptible to critically or highly important antibiotics like daptomycin, teicoplanin, tigecycline, and vancomycin, the presence of GDRs in MDR-plasmids is a concern since antimicrobials commonly used in livestock could co-select and confer resistance to critically important antimicrobials not used in food-producing animals.

Campylobacter jejuni and Campylobacter coli are important foodborne zoonotic pathogens and cause for concern due to the increasing trend in antimicrobial resistance. A long-run surveillance study was conducted in animals from different age groups in five dairy cattle farms to investigate the within-farm diversity and transmission dynamics of resistant Campylobacter throughout time. The resistance phenotype of the circulating isolates (170 C. jejuni and 37 C. coli) was determined by broth microdilution and a selection of 56 isolates were whole genome sequenced using the Oxford-Nanopore long-fragment sequencing technology resulting in completely resolved and circularized genomes (both chromosomes and plasmids). C. jejuni was isolated from all farms while C. coli was isolated from only two farms, but resistance rates were higher in C. coli than in C. jejuni and in calves than in adult animals. Some genotypes (e.g. ST-48, gyrA_T86I/tet(O)/bla_OXA-61 in farm F1; ST-12000, aadE-Cc/tet(O)/bla_OXA-489 in F4) persisted throughout the study while others were only sporadically detected. Acquisition of extracellular genes from other isolates and intracellular mutational events were identified as the processes that led to the emergence of the resistant genotypes that spread within the herds. Monitoring with Oxford Nanopore Technologies sequencing helped to decipher the complex molecular epidemiology underlying the within-farm dissemination of resistant Campylobacter.

Anaplasma species are obligate intracellular rickettsial vector-borne pathogens that impose economic constraints on animal breeders and threaten human health. Anaplasma ovis and Anaplasma phagocytophilum infect sheep and goats worldwide. A duplex PCR targeting the msp2 and msp4 genes of A. phagocytophilum and A. ovis, respectively, was developed to analyze the field blood samples collected from sheep and goats. A total of 263 apparently healthy small ruminants from 16 randomly selected flocks situated in 3 bioclimatic zones in Tunisia were analyzed for Anaplasma infections. Anaplasma spp. was detected in 78.3% (95% confidence interval (CI): 72.8–83.1) of the analyzed animals. The prevalence of A. ovis in sheep (80.4%) and goats (70.3%) was higher than that of A. phagocytophilum (7.0% in sheep and 1.6% in goats). Using an inexpensive, specific, and rapid duplex PCR assay, we provide, to the best of our knowledge, the first molecular evidence for the presence of A. phagocytophilum in small ruminants in Tunisia. A. phagocytophilum generally presented as a co-infection with A. ovis. This study provides important data to understand the epidemiology of anaplasmosis in small ruminants, and highlights the risk of contracting the infection upon tick exposure.

NDM-producing Enterobacteriaceae have increased worldwide in human infections, but are still rare in food-producing animals. ¹ The only NDM type described in cattle so far was NDM-5, ^2–4 always carried by IncX plasmids. To the best of our knowledge, this study represents the first description of a bla_NDM-1 gene in Escherichia coli isolated from cattle, carried in an IncC plasmid.

We report here the draft genome sequence of an extended-spectrum β-lactamase (ESBL)-producing Escherichia species isolated from rectal feces collected from beef cattle in northern Spain. Analysis of the draft genome identified the strain as a member of the newly described species Escherichia marmotae .

In order to estimate herd-level prevalence of ESBL-/AmpC- and carbapenemase-producing commensal Escherichia coli in ruminants in the Basque Country (Northern Spain), a cross-sectional survey was conducted in 2014–2016 in 300 herds using selective isolation. ESBL-/AmpC-producing E. coli were isolated in 32.9% of dairy cattle herds, 9.6% of beef cattle herds and 7.0% of sheep flocks. No carbapenemase-producing E. coli were isolated. Phenotypic antimicrobial susceptibility determined by broth microdilution using EUCAST epidemiological cut-off values identified widespread co-resistance to extended spectrum cephalosporins and other antimicrobials (110/135 isolates), particularly tetracycline, sulfamethoxazole, trimethoprim, and ciprofloxacin. All isolates were susceptible to tigecycline, imipenem, meropenem and colistin. The genome of 66 isolates was sequenced using Illumina NovaSeq6000 and screened for antimicrobial resistance determinants against ResFinder and PointFinder. Plasmid/chromosomal location of resistance genes was predicted with PlasFlow and plasmid replicons were identified using PlasmidFinder. Fifty-two acquired resistance genes and point mutations in another four genes that coded for resistance to eleven antimicrobial classes were identified. Fifty-five genomes carried ESBL-encoding genes, bla_CTX-M-14 being the most common, and 11 carried determinants of the AmpC phenotype, mostly bla_CMY-2 gene. Additionally, genes coding for β-lactamases of the CTX-M group 9 were detected as well as the sporadic presence of bla_SHV-12, bla_CMY-4, and a point mutation in the ampC promoter. Only a bovine isolate co-harbored more than one ESBL/AmpC genetic determinant (bla_CTX-M-14 and a mutation in the ampC promoter), confirming its ESBL+AmpC phenotype. Most ESBL/AmpC genes were located in IncI1 plasmids, which also carried a great variety of other AMR genes. Importance: Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC) producing E. coli have emerged in recent years as one of the fastest spreading antimicrobial resistance determinants in humans and food-producing animals, becoming a concern for animal and public health. This study provided an insight into the prevalence of cefotaxime-resistant E. coli in cattle and sheep in the Basque Country and the associated genetic determinants of antimicrobial resistance. These constituted an important contribution to the limited repository of such data for cattle in the region and for sheep worldwide. Antimicrobial susceptibility testing by phenotypic and molecular methods is key in surveillance programs to enhance early detection of resistance development, monitor resistance trends and provide guidance to clinicians in selecting the adequate therapy.

A cross-sectional survey was conducted in 2014–2016 in 301 ruminant herds to estimate C. jejuni and C. coli prevalence, and investigate their susceptibility to antimicrobials. Risk of shedding C. jejuni was higher in cattle than sheep (81.2% vs. 45.2%; OR_adj = 5.22, p < 0.001), whereas risk of shedding C. coli was higher in sheep than in cattle (19.1% vs. 11.3%; OR_adj = 1.71, p = 0.128). Susceptibility to six antimicrobials was determined by broth microdilution using European Committee for Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off values. C. coli exhibited higher resistance (94.1%, 32/34) than C. jejuni (65.1%, 71/109), and resistance was more widespread in isolates from dairy cattle than beef cattle or sheep. Compared to results obtained 10-years earlier (2003–2005) in a similar survey, an increase in fluoroquinolone-resistance was observed in C. jejuni from beef cattle (32.0% to 61.9%; OR = 3.45, p = 0.020), and a decrease in tetracycline-resistance in C. jejuni from dairy cattle (75.0% to 43.2%; OR = 0.25, p = 0.026). Resistance to macrolides remained stable at low rates and restricted to C. coli from dairy cattle, with all macrolide-resistant C. coli showing a pattern of pan-resistance. Presence of the single nucleotide polymorphisms (SNPs) associated to quinolone and macrolide resistance was confirmed in all phenotypically resistant isolates. The increase in fluoroquinolone resistance is worrisome but susceptibility to macrolides is reassuring.

Campylobacter is the main cause of gastroenteritis in humans in industrialized countries, and poultry is its principal reservoir and source of human infections. Dietary supplementation of broiler feed with additives could improve productive performance and elicit health benefits that might reduce Campylobacter contamination during primary production. The aim of this study was to assess the effect of dietary supplementation with whey (a prebiotic) and calcium butyrate (a salt of a short-chain fatty acid) on productive traits, duodenal histological integrity, and Campylobacter colonization and dissemination in broiler chickens during the 42-day rearing period. Six hundred one-day-old Ross-308 chickens were placed into 20 ground pens and assigned to one of 4 corn/soybean-based dietary treatments (5 replicates of 30 chicks per treatment) following a randomized complete block design: 1) basal diet with no supplementation as the control, 2) diet supplemented with 6% dry whey powder, 3) diet containing 0.1% coated calcium butyrate, and 4) diet containing 6% whey and 0.1% calcium butyrate. At age 15 days, 6 chickens per pen were experimentally inoculated with Campylobacter jejuni. The results showed that supplementation of the corn/soybean-based diet with 6% whey alone or, preferably, in combination with 0.1% coated calcium butyrate improved growth and feed efficiency, had a beneficial effect on duodenal villus integrity, and decreased mortality. These favourable effects were particularly significant during the starter period. Six days after oral challenge, Campylobacter was widespread in the flock, and the birds remained positive until the end of the rearing period. Although Campylobacter was not isolated from environmental samples, it was detected by real-time polymerase chain reaction (PCR) in dust, air filters, and drinkers while birds shed culturable C. jejuni cells. No differences (p > 0.050) in colonization or shedding levels that could be attributed to the diet were observed during the assay. Beneficial effects on performance and intestinal health were observed, particularly during the starter period, when chickens were fed a diet supplemented with both whey and coated calcium butyrate. However, none of the tested diets provided the chicks any differential degree of protection against Campylobacter infection.

Tick-borne diseases caused by Anaplasma species put serious constraints on the health and production of domestic cattle in tropical and sub-tropical regions. After recovering from a primary infection, cattle typically become persistent carriers of pathogens and play a critical role in the epidemiology of the disease, acting as reservoirs of the Anaplasma spp. In this study a duplex PCR assay was used for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle using two primer pairs targeting msp4 and msp2 genes, respectively. We used this method to analyze DNA preparations derived from 328 blood cattle samples that were collected from 80 farms distributed among Tunisia’s four bioclimatic zones. The prevalence of the A. marginale infection (24.7 %) was significantly higher and more widespread (in all bioclimatic areas) than that of A. phagocytophilum (0.6 %), which was found in a mixed infection with A. marginale. The duplex PCR assay used proved to be a rapid, specific and inexpensive mean for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle blood. It allowed us to report the identification of A. phagocytophilum for the first time in cattle in Tunisia and confirm the presence of A. marginale in cattle from several geographical areas of the country. Further epidemiological studies undertaken using this assay will help improve the surveillance of the associated diseases in the regions where they are endemic.