Effect of amoxicillin-clavulanic acid on clinical scores, intestinal microbiome, and amoxicillin-resistant Escherichia coli in dogs with uncomplicated acute diarrhea.

BACKGROUND
Despite limited evidence of efficacy, antibiotic treatment is still frequently prescribed in dogs with uncomplicated acute diarrhea (AD).


OBJECTIVE
To assess whether amoxicillin-clavulanic acid has a clinical benefit, an effect on the fecal microbiome, and the proportion of amoxicillin-resistant Escherichia coli in dogs with AD.


ANIMALS
Sixteen dogs with AD of <3 days duration.


METHODS
Prospective, placebo-controlled, double-blinded study. Clinical scores were compared between client-owned dogs randomly assigned to an antibiotic (AG) or a placebo (PG) group. The intestinal microbiome was analyzed using quantitative PCR assays. Amoxicillin-resistant fecal E. coli were assessed semiquantitatively with microbiological methods.


RESULTS
There was no difference in clinical recovery between treated dogs or controls (CADS index day 10: AG group median: 2 (range: 1-3; CI [1.4; 2.6]); PG group median: 1.6 (range: 1-3; CI [1.1; 2.4]); P > .99). All dogs gained normal clinical scores (CADS index ≤3) after 1 to 6 days (median 2 days) after presentation. There was no significant difference in the fecal dysbiosis index (during treatment: AG mean -2.6 (SD 3.0; CI [-5.1; 0.0]); PG mean -0.8 (SD 4.0; CI [-4.2; 2.5]; P > .99) or its bacterial taxa. The proportion of resistant fecal E. coli increased (to median: 100%; range: 35%-100%) during treatment with amoxicillin-clavulanic acid and was still increased (median: 10%; range 2%-67%) 3 weeks after treatment, both of which were significantly higher proportions than in the placebo group for both time points (during treatment AG median 100% versus PG median 0.2% (P < .001); after treatment AG median 10% versus PG median 0.0% (P = .002)).


CONCLUSIONS AND CLINICAL IMPORTANCE
Our study suggests that treatment with amoxicillin-clavulanic acid confers no clinical benefit to dogs with AD, but predisposes the development of amoxicillin-resistant E. coli, which persist for as long as 3 weeks after treatment. These findings support international guideline recommendations that dogs with diarrhea should not be treated with antimicrobials unless there are signs of sepsis.

significantly higher proportions than in the placebo group for both time points (during treatment AG median 100% versus PG median 0.2% (P < .001); after treatment AG median 10% versus PG median 0.0% (P = .002)).

Conclusions and Clinical Importance:
Our study suggests that treatment with amoxicillin-clavulanic acid confers no clinical benefit to dogs with AD, but predisposes the development of amoxicillin-resistant E. coli, which persist for as long as 3 weeks after treatment. These findings support international guideline recommendations that dogs with diarrhea should not be treated with antimicrobials unless there are signs of sepsis. tions. [2][3][4][5][6] In many cases the etiology cannot be identified. This is usually not a problem, because clinical signs typically resolve spontaneously and usually do not recur. 2,4,[7][8][9] International guidelines recommend that in dogs with diarrhea, antimicrobials should only be administered to dogs manifesting systemic signs of illness. [10][11][12][13][14] Despite these recommendations, it is common that dogs with AD receive an untargeted, short-term antibiotic course as first-line medication. Two surveys including 11 060 and 371 dogs performed in Europe showed that between 63% and 71% of dogs with AD were treated with antimicrobials, respectively. 15,16 Our study group previously evaluated the effect of amoxicillin-clavulanic acid in dogs with acute hemorrhagic diarrhea syndrome (AHRD) without signs of sepsis, and were unable to show a clinical benefit of antibiotic therapy. 17,18 A second study in dogs with AHDS revealed that the additional application of metronidazole to amoxicillin-clavulanic acid did not improve the clinical outcome. 18 Antibiotic treatment can lead to various negative short-term effects, such as vomiting, diarrhea, and anorexia. 19,20 Data from human medicine suggest that dysbiosis induced by antimicrobials is associated with an increased risk for developing asthma, postinfectious irritable bowel syndrome and chronic enteropathies such as Crohn's disease. [21][22][23][24][25] Antibiotics cause prolonged intestinal dysbiosis and lead to changes in the microbial metabolism pathways in healthy dogs and humans. [26][27][28][29] Disruption of the intestinal microbiota as a consequence of antibiotic use can lead to life-threatening Clostridiodes difficile infections in humans. 30 Furthermore, antibiotic treatment leads to advantages in growth for resistant bacteria and provokes the development of new resistance mechanisms. [31][32][33][34][35] Although antibiotics have been routinely used in dogs with uncomplicated AD over decades, evidence-based studies documenting any clinical benefit of antibiotic treatment in dogs are sparse 36

| Sample collection
Naturally passed fecal samples from each dog were collected by the clinician on day 0 before starting the treatment, and by the owner on

| Quantitative PCR (qPCR)
For chosen bacterial taxa (ie, Faecalibacterium spp., Turicibacter spp., Streptococcus spp., E. coli, Blautia spp., Fusobacterium spp., and Clostridium hiranonis) and total bacteria, which are known to be altered in dogs with gastrointestinal disease, individual qPCR assays were performed and results used to calculate the recently described dysbiosis index (DI). 37 The method, including the oligonucleotide sequence of the primers and the annealing temperatures were described elsewhere previously. 37

| Evaluation of the proportion of resistant fecal E. coli
One gram of feces was placed into a test tube and mixed with 9 mL of phosphate buffered saline (PBS; pH = 7.0). The mixture was homoge-

| Statistical analyses
Power analysis determined that in order to detect a clinically relevant difference of 2 points in the CADS-Index at day 3 between the AG and the PG, at least 8 dogs per group had to be included (with an estimated SD of 1.5, power of 80% and P < .05).
Statistical analyses were conducted with GraphPad Prism (GraphPad Prism c7.0, GraphPad Software, San Diego, California).  Regarding presence and frequency of vomiting, fecal frequency, activity, and appetite there was no significant difference between groups on all days of the study period.

| Microbiome analysis
There was no statistically significant difference of the DI (day 0 AG investigated taxa between the groups at any time point (Figure 2, Table 3). An increased DI (> 2) was found in 7 dogs (AG colonies rose to 100% in the AG group (range 35%-100%), in the PG group the median was 0.2% (range 0%-10%, P < .001). On day 30, 3 weeks after discontinuing antibiotic treatment, there was still a significant higher percentage of resistant E. coli in the AG group (median 10%; range 2%-67%) than in the PG group (median 0%; range 0%-4%; P = .002; Figure 4).

| DISCUSSION
In this prospective, double-blinded, placebo-controlled study, clinical improvement of dogs with acute uncomplicated diarrhea was compared between dogs only treated with symptomatic treatment and those also treated with amoxicillin-clavulanic acid. There was no statistically significant difference in the clinical course between groups at any time point.
Oral treatment with amoxicillin-clavulanic acid led to a significant increase of resistant E. coli isolates, but based on the DI, did not result in a significantly more prolonged dysbiosis compared with the PG.
The absence of a beneficial clinical effect of amoxicillin-clavulanic acid treatment in dogs with uncomplicated AD is an important finding for clinicians, who routinely manage these cases on a daily basis.
Amoxicillin-clavulanic acid was chosen because beta-lactam antibiotics are the most frequently used antimicrobials in gastrointestinal disease in dogs and cats. 1,16 It is estimated that currently more than half of dogs with AD are Moreover, in the present study the treatment with amoxicillinclavulanic acid did not result in significant alterations in the DI or its included taxa during as well as 3 weeks after the treatment. In contrast, another study described a significant alteration in the intestinal microbiome in healthy dogs receiving amoxicillin. [26][27][28] This discrepancy could partially be explained because the cited study was performed in healthy dogs, no control group for comparison of intestinal microbiota changes was used, and a huge interpatient variability was seen.
Recent investigations showed a profound effect of metronidazole and tylosin on the microbiota. Tylosin significantly increased the DI during treatment, and the abundance of Faecalibacterium was significantly decreased after treatment. 46  C. difficile is known to induce pseudomembranous colitis and is associated with significant morbidity and mortality in humans. 53 Several studies showed that antibiotic use and more specifically the application of penicillin, is a major risk factor for the development of C. difficile infections. 54 66,67 This emphasizes once more the importance of prudent antimicrobial usage in order to prevent spread of antibiotic resistance.
There are limitations of the study. First of all, owners gave the treatments at home. Although owners were trained in administering the capsules according to the schedule and to report if capsule administration was not possible, it cannot be completely ruled out that on some occasions, treatments were not administered. Moreover, clinical disease activity of dogs was scored by the owner themselves.
Although some variables included in scores that can be objectively assessed (eg, defecation frequency, fecal consistency based on fecal scoring system), some variables (eg, activity) are relatively subjective.
Clinical impression also depends on the time spent with the dog, which differs between owners. Moreover, all enrolled dogs are from the same geographical region, which makes generalization impossible.
Furthermore, complete fecal microbiota sequencing was not performed. Therefore, for the purpose of the microbiome analysis it cannot be ruled out that a different method would have been able to detect changes in the composition of the intestinal microbiome between dogs treated with and without amoxicillin-clavulanic acid.
No specific mechanism of resistance was investigated in this study.
The main limitation of this study is the small number of dogs in both treatment groups. Sample-size calculation was based on clinician's opinion, because comparable studies were lacking at the time when the study-protocol was designed. It is impossible to make clear recom-