3.2. Antimicrobial Susceptibility
The resistance of Escherichia coli against trimethoprim (p = 0.0006), chloramphenicol (p = 0.012) and tetracycline (p = 0.03) increased from D0 to D3. However, E. coli resistance to other antibiotics was not different after exposure to semen extenders. Furthermore, there was no association between exposure to semen extenders and the resistance of Staphylococcus simulans and Streptococcus equisimilis (p > 0.05).
Resistance to at least 1 of 14 antibiotics was found in 284 of 520 E. coli
isolates. Twenty-nine isolates were multidrug-resistant (resistant to at least three classes of antibiotics). There was a significant increase in resistance to trimethoprim, chloramphenicol and tetracycline after exposure to semen extenders with antibiotics. However, resistance was also found against sulfamethoxazole, ampicillin, azithromycin and tigecycline, but differences between time points were not significant. All E. coli
isolates were susceptible to cefotaxime, ceftazidime, ciprofloxacin, colistin, gentamicin, meropenem and nalidixic acid (Table 2
No correlation was found between the resistance of Streptococcus dysgalactiae
before and after exposure to semen extenders with antibiotics. Various Streptococcus dysgalactiae
isolates were resistant to three of eight antibiotics, including tetracycline, erythromycin and nitrofurantoin. All Streptococcus dysgalactiae
isolates were susceptible to cefalothin, clindamycin, oxacillin, penicillin and trimethoprim/sulfamethoxazole. For some antibiotics, cefoxitin, enrofloxacin, fusidic acid and gentamicin, sensitivity or resistance could not be assessed since no ECOFFs were mentioned in EUCAST (Table 3
All Enterococcus faecalis
isolates were susceptible to the six tested antibiotics (ampicillin, ciprofloxacin, gentamicin, linezolid, teicoplanin and tigecycline). For some antibiotics (chloramphenicol, daptomycin, erythromycin, quinupristin/dalfopristin, tetracycline and vancomycin), sensitivity or resistance could not be assessed since no ECOFFs were given in the EUCAST (Table 4
). All tested Streptococcus equi
isolates were susceptible to clindamycin, erythromycin, nitrofurantoin, oxacillin, penicillin, tetracycline and trimethoprim/sulfamethoxazole. For some antibiotics, cefalotin, cefoxitin, enrofloxacin, fusidic acid and gentamicin sensitivity or resistance could not be assessed since no ECOFFs were given in the EUCAST (Table 5
Resistance to at least one of seven antibiotics was detected in different Staphylococcus simulans
isolates, including oxacillin (25%), penicillin (50%), fusidic acid (50%) and trimethoprim/sulfamethoxazole (50%). One isolate was multidrug resistant. There was no association between exposure to antibiotics via artificial insemination and change in resistance. All Staphylococcus simulans
isolates were susceptible to erythromycin, gentamicin and tetracycline. For some antibiotics, cefalotin, cefoxitin, clindamycin, enrofloxacin and nitrofurantoin, sensitivity or resistance could not be assessed since no ECOFFs were given in the EUCAST (Table 6
3.3. Whole-Genome Sequencing
Whole-Genome Sequencing was used to decide whether E. coli, Streptococcus equisimilis and Staphylococcus simulans isolates of different antimicrobial susceptibility from the same mares sampled at both time points were likely to be of the same strain or not and if phenotypic resistance was associated with genes for resistance.
One hundred twenty-seven E. coli
isolates from inseminated mares before (D0) and after (D3) insemination, were allocated into fourteen clusters by cgMLST analysis (Figure 1
). There was, at most, a one-allele difference between isolates within each cluster, except for Cluster 2 and 13 where there was a three-allele difference. Therefore, isolates within each cluster were highly genetically related, i.e. the same strain. Nine clusters consisted of isolates from one mare (Cluster 3, 6, 7, 8, 9, 10, 11 and 14), whereas five clusters consisted of isolates from 2 to 3 mares (Cluster 1, 2, 4 5 and 13 (Figure 1
)). The finding of isolates from different mares in the same clusters suggests a possible spread within the herd.
Twenty-six Streptococcus equisimilis
isolates from inseminated mares before (D0) and after (D3) insemination, were subjected to cgMLST analysis, and a minimum spanning tree (MST) was generated (Figure 2
a). There were 18 allelic differences between 12 isolates from Horse H and 3 isolates from Horse J, but since the developed cgMLST scheme lacked a validated clustering distance threshold, it was not possible to determine whether the isolates belonged to 2 different clusters or 1 and the same (Figure 2
a). Regardless, there were no allelic differences between isolates from each horse in this case; hence, they were of the same strain. Eleven isolates from Horse I and J clustered together with a maximum of two allelic differences, and even if the developed cgMLST scheme lacked a validated clustering distance threshold, it is likely that those isolates were genetically closely related; i.e. they were the same strain since the differences were very few (Figure 2
a). The finding of genetically related isolates in two mares (Horse I and J) indicates a possible spread within the herd, as seen in E. coli
Four Staphylococcus simulans
isolates from an inseminated mare (Horse K) before (D0) and after (D3) insemination, were subjected to cgMLST analysis, and a minimum spanning tree (MST) was generated (Figure 2
b). There were no allelic differences between isolates P868 and PM194 and P869 and PM193, respectively; hence, isolates within each pair were genetically closely related, i.e. the same strain. Although no defined cluster distance threshold was available for this cgMLST scheme, the allelic distance between the two pairs was very large, and it is therefore unlikely that they are genetically closely related (Figure 2
Twenty-nine of the E. coli isolates were multiple drug resistant (MDR); twenty of these isolates belonged to Cluster 2 (Horse C and G), with one isolate each belonging to Cluster 4 (Horse B), Cluster 6 (Horse E), Cluster 7 (Horse D) and Cluster 14 (Horse B).
Eighteen of the Streptococcus equisimilis isolates were phenotypically resistant to tetracycline; eight clustered together from Horse H; ten clustered with isolates from Horse I and J. One isolate from Horse H was phenotypically resistant to erythromycin. One isolate was phenotypically resistant to nitrofurantoin, and it clustered together with isolates from Horse I and J.
Two isolates (P868 and PM194), which clustered together, were phenotypically resistant to fusidic acid. Two other isolates that clustered together (P869 and PM193) were phenotypically resistant to penicillin and trimethoprim/sulfamethoxazole. One isolate (P869) was phenotypically resistant to oxacillin.
The phenotypic resistance was compared between isolates from individual mares before and after the exposure to antibiotics within the same cluster, i.e. isolates of the same strain (Table 7
and Table 8
). For E. coli,
increased resistance to sulfamethoxazole (Cluster 1), trimethoprim (Cluster 2 and 10), chloramphenicol (Cluster 2, 6 and 14) and tetracycline (Cluster 6) was found.
All sequenced Escherichia coli isolates were resistant to sulfamethoxazole both phenotypically and genotypically; 6 isolates had the sul1 gene, and 26 isolates had the sul2 gene, which are responsible for sulfamethoxazole resistance. However, no resistance genes were found in 95 of the isolates. Thirty-two E. coli isolates were phenotypically resistant to trimethoprim; twenty-six of these isolates had dfrA1; seventeen isolates had dfrA14; no resistance genes were found in two isolates. Ten E. coli isolates were phenotypically resistant to tetracycline; six of these isolates had tet(A); one isolate had marR_S3N; no resistance genes were found in the other three isolates. All 32 E. coli isolates that were phenotypically resistant to chloramphenicol had mdf(A). One E. coli isolate phenotypically resistant to tigecycline had mdf(A). Of the seven E. coli isolates phenotypically resistant to ampicillin, six isolates had blaEC-5/blaTEM-1 and blaTEM-1B; one isolate had blaEC.
The gene marR_S3N was found in twenty-seven E. coli isolates susceptible to ciprofloxacin, chloramphenicol and tigecycline and twenty-six isolates susceptible to tetracycline. The gene blaEC was found in 119 isolates susceptible to meropenem, cefotaxime and ceftazidime and 118 isolates susceptible to ampicillin. The gene blaEC-5/blaTEM-1 was found in six isolates susceptible to meropenem, cefotaxime and ceftazidime.
The gene catB3 was found in six E. coli isolates susceptible to chloramphenicol. The gene tet(A) was found in six isolates susceptible to tigecycline. The gene aadA5 was found in six isolates susceptible to gentamicin. The gene aph(6)-ld was found in 26 isolates susceptible to gentamicin. In addition, the gene mdf(A), conferring resistance to broad-spectrum drugs via an efflux pump, was found in 92 isolates.
Five Streptococcus equisimilis isolates had lsaC as a resistance gene; one of these isolates had phenotypical resistance to erythromycin. Eighteen isolates phenotypically resistant to tetracycline were not shown to have resistance genes. Furthermore, one isolate, which was phenotypically resistant to nitrofurantoin, did not have resistance genes.
Two Staphylococcus simulans isolates had the blaZ resistance gene responsible for phenotypical resistance to penicillin and oxacillin. However, two isolates phenotypically resistant to fusidic acid and two isolates phenotypically resistant to trimethoprim/sulfamethoxazole did not have any resistance genes.