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An easily implementable strategy to reduce treatment failures in severe bacterial infections is to combine already available antibiotics. However, most in vitro combination assays are performed by exposing standard bacterial inocula to constant concentrations of antibiotics over less than 24h, which can be poorly representative of clinical situations. The aim of this study was to assess the ability of static and dynamic in vitro Time-Kill Studies (TKS) to identify the potential benefits of an antibiotic combination (here, amikacin and vancomycin) on two different inoculum sizes of two S. aureus strains. In the static TKS (sTKS), performed by exposing both strains over 24h to constant antibiotic concentrations, the activity of the two drugs combined was not significantly different the better drug used alone. However, the dynamic TKS (dTKS) performed over 5 days by exposing one strain to fluctuating concentrations representative of those observed in patients showed that, with the large inoculum, the activities of the drugs, used alone or in combination, significantly differed over time. Vancomycin did not kill bacteria, amikacin led to bacterial regrowth whereas the combination progressively decreased the bacterial load. Thus, dTKS revealed an enhanced effect of the combination on a large inoculum not observed in sTKS. The discrepancy between the sTKS and dTKS results highlights that the assessment of the efficacy of a combination for severe infections associated with a high bacterial load could be demanding. These situations probably require the implementation of dynamic assays over the entire expected treatment duration rather than the sole static assays performed with steady drug concentrations over 24h.

An antibiotic susceptibility monitoring programme was conducted from 2004 to 2010, resulting in a collection of 143 Escherichia coli cultured from bovine faecal samples (diarrhoea) and milk-aliquots (mastitis). The isolates were subjected to whole-genome sequencing and were distributed in phylogroups A, B1, B2, C, D, E, and G with no correlation for particular genotypes with pathotypes. In fact, the population structure showed that the strains belonging to the different phylogroups matched broadly to ST complexes; however, the isolates are randomly associated with the diseases, highlighting the necessity to investigate the virulence factors more accurately in order to identify the mechanisms by which they cause disease. The antimicrobial resistance was assessed phenotypically, confirming the genomic prediction on three isolates that were resistant to colistin, although one isolate was positive for the presence of the gene mcr-1 but susceptible to colistin. To further characterise the genomic context, the four strains were sequenced by using a single-molecule long read approach. Genetic analyses indicated that these four isolates harboured complex and diverse plasmids encoding not only antibiotic resistant genes (including mcr-1 and bla) but also virulence genes (siderophore, ColV, T4SS). A detailed description of the plasmids of these four E. coli strains, which are linked to bovine mastitis and diarrhoea, is presented for the first time along with the characterisation of the predicted antibiotic resistance genes. The study highlighted the diversity of incompatibility types encoding complex antibiotic resistance elements such as Tn6330, ISEcp1, Tn6029, and IS5075. The mcr-1 resistance determinant was identified in IncHI2 plasmids pCFS3273-1 and pCFS3292-1, thus providing some of the earliest examples of mcr-1 reported in Europe, and these sequences may be a representative of the early mcr-1 plasmidome characterisation in the EU/EEA.

The animal reservoir of Enterobacterales producing Extended-Spectrum-β-Lactamases (ESBL) and plasmid-borne cephalosporinases (pAmpC) is a global concern. Using genome data, we analyzed a population of Escherichia coli and Salmonella species resistant to third-generation cephalosporins (3GC-R) recovered from healthy food animals (HA) and diseased food animals (DA) across Europe. Among the isolates collected from HA (n = 4,498) and DA (n = 833) in up to twelve European countries, 62 (1.4%) and 45 (5.4%) were 3GC-R, respectively. The genomes of these 3GC-R 107 isolates were sequenced to identify bla ESBL and bla AmpC , sequence types (STs), virulence-associated genes, and Salmonella serovars. We also assessed their population structure using core genome multilocus sequence typing. The 78 3GC-R Escherichia coli originated from poultry (n = 27), swine (n = 26), and cattle (n = 25). Almost all (n = 77; 98.7%) harbored at least one bla ESBL or bla AmpC , with bla CTX-M-1 predominating. We identified 51 STs, with ST10 and ST101 being the most frequent. The population of 3GC-R E . coli was polyclonal. The 29 3GC-R Salmonella spp. were mostly retrieved from healthy broiler (96.5%). bla CMY-2 dominated in this population. We found two clusters of CMY-2-producing Salmonella spp. in Germany: one with 15 isolates of S . Heidelberg isolates and another with six S . Minnesota, all of them with bla CMY-2 . Our results confirm the low prevalence of 3GC-R E . coli and Salmonella spp. in HA and DA. bla CTX-M-1 was dominating in a highly diverse population of E . coli . 3GC-R E . coli isolated from HA and DA were genetically unrelated, with high clonal diversity suggesting multiple origins of contamination. This contrasted with the clonal population of 3GC-R Salmonella spp. in which bla CMY-2 dominated through two dominant serovars in this collection.

Bacterial urinary tract infections (UTIs) occur frequently in companion animals and are often treated with antibiotics. However, antimicrobial resistance can severely hamper treatment success. Therefore, antimicrobial susceptibility monitoring is key. UTI isolates were obtained from dogs and cats in two collection periods (ComPath II: 2013–2014 and ComPath III: 2017–2018) as part of CEESA’s ComPath programme. Susceptibility testing of the UTI isolates (2021 in total) was carried out at one central laboratory using agar and broth dilution methodology as recommended by the Clinical and Laboratory Standards Institute. Escherichia coli was the most frequently isolated bacterium in UTI in both dogs (46.9%, 43.1%) and cats (61.2%, 48.3%) across ComPath II and ComPath III, respectively. The percentage of resistance in E. coli was low (<10%) across both programmes in both dogs and cats except for trimethoprim-sulfamethoxazole (dogs ComPath III: 12.9%; cats ComPath II: 13.0%) and enrofloxacin (10.5%), marbofloxacin (11.4%), and doxycycline (98.8%) for dogs in ComPath III. Three (7.5%) of the 40 isolated S. aureus bacteria in total were MRSA and harboured mecA. The level of multidrug resistance (MDR) was generally low and ranged from 0.0% for feline coagulase-negative Staphylococcus spp. to 11.7% for canine Proteus spp., except for a peak of MDR observed in canine Klebsiella isolates from ComPath II (36.7%). Overall, antimicrobial resistance for most canine and feline UTI pathogens isolated during the ComPath II and ComPath III programmes was low (1–10%) to moderate (10–20%).

We investigated the antimicrobial activity of a fluoroquinolone against two genetically close bacterial species belonging to the Pasteurellaceae family. Time-kill experiments were used to measure the in vitro activity of marbofloxacin against two strains of Mannheimia haemolytica and Pasteurella multocida with similar MICs. We observed that marbofloxacin was equally potent against 105 CFU/mL inocula M. haemolytica and P. multocida. However, an inoculum effect was observed with P. multocida, meaning that marbofloxacin activity was decreased against a 108 CFU/mL inoculum, whereas no inoculum effect was observed with M. haemolytica. Marbofloxacin activity was also tested in a lung infection model with immunocompromised mice intratracheally infected with 109 CFU of each bacteria. At the same dose, the clinical and bacteriological outcomes were much better for mice infected with M. haemolytica than for those infected with P. multocida. Moreover, bacteriological eradication was obtained with a lower marbofloxacin dose for mice infected with M. haemolytica. Our results suggest that the differential in vivo marbofloxacin efficacy observed with the two bacterial species of similar MIC could be explained by a differential inoculum effect. Consequently, MICs determined on 105 CFU inocula were not predictive of the differences in antibiotic efficacies against high bacterial inocula of closely related bacterial strains. These results could stimulate further investigations on bacterial species-specific antibiotic doses in a clinical setting.

The therapeutic efficacy of an early treatment protocol with an infection-stage adjusted fluoroquinolone regimen was evaluated in a field study on young bulls (YBs) presenting signs of bovine respiratory disease (BRD). A total of 195 YB (Charolais, Limousin, and Rouge-des-Prés breeds) from 6 farms implementing or not prophylactic antimicrobial treatments (PROPHY or absence) were randomly assigned to 1 of 2 experiment groups based on time of detection of BRD and first-line marbofloxacin regimen, early adjusted dose [Early 2 (E2)] or late standard dose [Late 10 (L10)]. Each YB was administered orally a reticulo-rumen bolus, allowing continuous monitoring of ruminal temperature. In the E2 group, YB presenting early signs of BRD, i.e., an increase in ruminal temperature over 40.2°C and persisting more than 12 h, confirmed by a clinical examination showing no or mild signs of BRD, were given 2 mg/kg of marbofloxacin. In the L10 group, YBs presenting moderate or severe signs of BRD at visual inspection, confirmed at clinical examination, were given 10 mg/kg of marbofloxacin. If needed, YBs were given a relapse treatment. The YBs were followed for 30 days. The proportions of first and relapse treatments were calculated, as well as the therapeutic efficacy at day 10. In the E2 group, the first-line treatments' proportion was significantly higher (  < 0.05), while the relapse treatments' proportion tended to be higher (  = 0.08), than in the L10 group. Evolution of clinical scores (CSs) of diseased YB was followed for 10 days. In both groups, CS and rectal temperature decreased significantly 24 h after treatment (  < 0.05). Treatment incidences (TI) representing antimicrobial consumption assessed on used daily doses (UDD) were calculated. Antimicrobial consumption of marbofloxacin and relapse treatments were not significantly different between the groups. These values were strongly influenced by the recourse to a prophylactic antimicrobial treatment, accounting for more than 90% of the antimicrobial amount in the herds implementing prophylaxis. The higher number of treatments in the groups treated on the basis of ruminal temperature monitoring, the accuracy of the detection method, and the necessary conditions to implement precision antimicrobial therapy in the field are discussed in this article.

Background Antimicrobial resistance (AMR) is a global concern impacting both humans, animals and their environment. The use of oral antimicrobials in livestock, particularly in pigs, has been identified as a driver in the selection of AMR bacteria. The aim of the present study was to evaluate the effects of a single intramuscular (IM) dose of marbofloxacin (8 mg/kg) on Enterobacteriaceae and E. coli populations, as well as on fluoroquinolone resistance within the fecal microbiota of pigs. Twenty healthy pigs, 60-days old, were divided into two groups: a treated group ( n  = 13) and a control group ( n  = 7) and were monitored over a 28-day experimental period. Fecal samples were collected from all animals for the isolation of E. coli and Salmonella strains. The minimum inhibitory concentration (MIC) of marbofloxacin for the isolates recovered on MacConkey agar supplemented with 1 or 4 µg/mL of marbofloxacin and for some generic E. coli isolates (recovered from MacConkey agar not supplemented with marbofloxacin) was determined using the broth microdilution method. Genomic DNA was extracted from the confirmed bacterial strains and sequenced using the Sanger method to identify mutations in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes. Results The single IM administration of marbofloxacin resulted in a significant decrease in Enterobacteriaceae and E. coli fecal populations from days 1 to 3 post- treatment. No Salmonella isolates were detected in either group, and no marbofloxacin-resistant E. coli isolates were identified. The MIC of the selected generic E. coli strains ( n  = 100) showed an increase to up to 0.5 µg/mL between days 1 and 3 post-treatment but remained below the clinical breakpoint of marbofloxacin resistance (4 µg/mL). Sequencing of these isolates revealed no mutations in gyrA and parC genes. Conclusions The present study showed that this dosing regimen of marbofloxacin significantly decreases the fecal shedding of Enterobacteriaceae and E. coli populations in pigs, while limiting the selection of marbofloxacin-resistant E. coli isolates. These findings warrant validation in sick pigs to support the selective use of this antibiotic solely in cases of clinical disease, thereby minimizing the reliance on conventional (metaphylactic) group treatments in pigs.

ComPath is a pan-European antimicrobial surveillance program collecting bacterial pathogens from dogs and cats not recently exposed to antimicrobials. We present minimum inhibitory concentration data obtained using Clinical and Laboratory Standards Institute methodology for 616 urinary tract infection (UTI) isolates collected between 2008 and 2010. In both dogs and cats, the most common pathogen was Escherichia coli (59.8% and 46.7%, respectively). Antimicrobial activity against E. coli in dogs and cats was similar with fluoroquinolone and trimethoprim/sulfamethoxazole susceptibility >90%. Ampicillin susceptibility was ∼80%. Staphylococcus intermedius Group isolates from dogs (67/437, 15.3%) had high antimicrobial susceptibility (>90%) toward beta-lactams, fluoroquinolones, and trimethoprim/sulfamethoxazole. Four canine isolates (6%) were oxacillin resistant, and harbored mecA . Proteus mirabilis from dogs (48/437, 11.0%) had high antimicrobial susceptibility (∼90%) to amoxicillin/clavulanic acid, enrofloxacin, and marbofloxacin and slightly lower susceptibility (∼80–85%) to ampicillin and orbifloxacin. Streptococcus canis isolates (35/437, 8.0%) from dogs were all susceptible to ampicillin and amoxicillin/clavulanic acid and >90% susceptible to marbofloxacin. Although resistance was not observed, high intermediate susceptibility was seen for both enrofloxacin (28.6%) and orbifloxacin (85.7%). Overall, antimicrobial in vitro activity appears to be high in UTI pathogens from dogs and cats with low multidrug resistance, although a lack of specific dog and cat breakpoints for important antimicrobials such as cefovecin, cephalexin, and ibafloxacin prevents analysis of susceptibility for these agents.

The study aimed to characterize beta-lactam resistance mechanisms of Enterobacteriaceae isolates recovered from diseased dogs and cats between 2008 and 2010 in a European surveillance program (ComPath I) for the antibiotic susceptibility of bacterial pathogens. A total of 608 non-duplicated Enterobacteriaceae isolates were obtained prior antibiotic treatment from diseased dogs ( n =464) and cats ( n =144). Among the 608 Enterobacteriaceae isolates, 22 presented a minimal inhibitory concentration against cefotaxime above EUCAST breakpoints of susceptibility. All the 22 isolates remained susceptible to carbapenems. Ten isolates were confirmed as extended-spectrum-beta-lactamase (ESBL) producers by PCR-sequencing of bla coding genes including 9 bla CTX-M (CTX-M-1, 14, 15, 32,…) and 1 bla TEM-52 and 12 were AmpC-producing isolates (10 plasmidic CMY-2 group and 2 isolates overexpressing their chromosomal AmpC). ESBLs and plasmid-mediated AmpC (pAmpC)-producing isolates were mainly recovered from dogs ( n =17) suffering from urinary tract infections ( n =13) and originated from eight different countries. ESBL-bearing plasmids were mostly associated with IncFII incompatibility groups while CMY-2 was predominantly associated with plasmid of the IncI1 group. ESBL/pAmpC-producing Escherichia coli belonged to phylogroup A ( n =5), B2 ( n =4), and D ( n =5). Multilocus sequence typing analysis revealed that among three CTX-M-15-producing E. coli , two belong to sequence type (ST) 131 and one to ST405. The presence of CTX-M-15 including on IncFII plasmids in E. coli ST131-B2 has also been described in isolates of human origin. This suggests the possibility of exchanges of these isolates from humans to companion animals or vice-versa.

The animal reservoir of Enterobacterales producing Extended-Spectrum-β-Lactamases (ESBL) and plasmid-borne cephalosporinases (pAmpC) is a global concern. Using genome data, we analyzed a population of Escherichia coli and Salmonella species resistant to third-generation cephalosporins (3GC-R) recovered from healthy food animals (HA) and diseased food animals (DA) across Europe. Among the isolates collected from HA (n = 4,498) and DA (n = 833) in up to twelve European countries, 62 (1.4%) and 45 (5.4%) were 3GC-R, respectively. The genomes of these 3GC-R 107 isolates were sequenced to identify blaESBL and blaAmpC, sequence types (STs), virulence-associated genes, and Salmonella serovars. We also assessed their population structure using core genome multilocus sequence typing. The 78 3GC-R Escherichia coli originated from poultry (n = 27), swine (n = 26), and cattle (n = 25). Almost all (n = 77; 98.7%) harbored at least one blaESBL or blaAmpC, with blaCTX-M-1 predominating. We identified 51 STs, with ST10 and ST101 being the most frequent. The population of 3GC-R E. coli was polyclonal. The 29 3GC-R Salmonella spp. were mostly retrieved from healthy broiler (96.5%). blaCMY-2 dominated in this population. We found two clusters of CMY-2-producing Salmonella spp. in Germany: one with 15 isolates of S. Heidelberg isolates and another with six S. Minnesota, all of them with blaCMY-2. Our results confirm the low prevalence of 3GC-R E. coli and Salmonella spp. in HA and DA. blaCTX-M-1 was dominating in a highly diverse population of E. coli. 3GC-R E.coli isolated from HA and DA were genetically unrelated, with high clonal diversity suggesting multiple origins of contamination. This contrasted with the clonal population of 3GC-R Salmonella spp. in which blaCMY-2 dominated through two dominant serovars in this collection.