Report: antimicrobial resistance in commensal Enterococcus spp. from poultry, pigs, cows and veal calves

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1 Veterinary and Agrochemical Research Centre Report: antimicrobial resistance in commensal Enterococcus spp. from poultry, pigs, cows and veal calves P. Butaye 1 Introduction Enterococci are regarded as general indicators for resistance amongst Gram positive bacteria, similarly to E. coli for the Gram negative bacteria. As with E. coli they have the advantage of being present in nearly all animal species, however this is frequently age dependent and the numbers of bacteria are smaller compared to E. coli, as reflected in lower isolation successes shown in different surveillance programmes. At the other hand, they are the most prevalent facultative aerobic Gram positive bacteria and as such most suitable for antimicrobial resistance surveillance. Because they are continuously present, they can also be used to follow up resistance evolution in time. Enterococci are a diverse group of bacteria. They can be dividend in species groups. The species mainly involved in surveillances are E. faecalis and E. faecium. Isolating and recognising these bacteria on enterococcal selective plates, as Slanetz and Bartley agar plates, is not always evident. Species belonging to the E. faecium group are difficult to separate and the species frequently co-isolated are E. hirae and E. durans. The use of a specific PCR only allows detecting a single species or a limited number of species when a multiplex PCR is used. There are no PCRs available allowing the unambiguous identification of the species of the E. faecium group. There exists however a well validated PCR technique allowing the identification of multiple species in one test. This technique is t-dna PCR, which is based on the amplification of the intergenic spacers between the genes encoding t-rna. Exact sizing of the obtained fragments by capillary electrophoresis and comparison with a database containing the profiles of the different bacterial species allows the unambiguous assignment to a species. Enterococci have been studied frequently in other countries. This will allow the comparison of antimicrobial resistances in enterococci from different geographical regions. The genetic background or resistance in this species is also quite well known allowing a scientific interpretation of the resistance data. 2 Materials and Methods 2.1 Sampling Samples from faecal material were taken from 4 animal categories: broiler chickens, pigs, bovines (for meat production) and veal calves. Samples were taken by samplers of the Belgian Food Agency Poultry Caecal content of broiler chickens was taken at slaughter together with the samples in the framework of Salmonella control programme. Caeca from 10 animals were collected and pooled. One sample originated from one farm.

2 2.1.2 Pig Pooled fresh faecal material of at least ten animals of approximately 6 months old was collected from slaughter pigs at the abattoir. One sample originated from one farm Bovines Pooled fresh faecal material was collected from the floor of barns harbouring bovines for meat production of less than 7 months of age. One sample originated from one farm Veal calves Pooled fresh faecal material was collected at the abattoir from veal calves of less than 7 months of age. 2.2 Isolation and identification As compared to 2011, methodology of isolation has been changed to increase the isolation success. At first faecal material was inoculated into a 7% NaCl supplemented BHI broth. One loopfull of this broth was then inoculated on Slanetz and Bartley agar plates and incubated at 37 C for hours at DGZ or ARSIA. Next to that, the number of samples taken was increased. Plates were then transferred to CODA-CERVA where the colonies with an enterococcal morphology were purified on blood agar plates and Slanetz and Bartley plates and incubated at 37 C for hours. Based on their growth aspects on both Slanetz and Bartley and blood agar plates, colonies were selected from identification. DNA was extracted using the alkalic extraction method and was stored at -20 C for further processing. t-dna intergenic spacer PCR was performed and obtained fragments were sized using capillary electrophoresis on a Beckman CEQ8000 sequencer (Baele et al., 1998). Obtained fragments were compared to the constructed database and strains were identified. Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae and Enterococcus durans were taken into account. 2.3 Susceptibility testing From a fresh culture on Columbia agar with 5% sheep blood, susceptibility was tested using a micro broth dilution method (Trek Diagnostics). To this end, 1 to 3 colonies were suspended in sterile distilled water to an optical density of 0.5 McFarland. Ten microliter of this suspension is inoculated in 11ml cation adjusted Mueller Hinton broth with TES buffer. Fifty microliter of the Mueller-Hinton broth with bacteria was brought on a micro-titer plate with the antimicrobials lyophilised, the NVL76 plate as produced by Trek Diagnostics, using the auto-inoculating system of Trek Diagnostics. The concentrations tested are indicated in table 1 (grey zones are the concentrations tested). Plates were incubated hours at 35 C and read. The Minimal Inhibitory Concentration (MIC) was defined as the lowest concentration by which no visible growth could be detected. MICs were semi- 2/75

3 automatically recorded by the Trek Vision system using the SWIN software. Results were automatically exported to an Excel file. Table 1 shows the antimicrobials tested and their abbreviations. Concentrations tested are shown in table Analysis of data Since isolation method was adapted, isolation successes from 2011 and 2012 were compared using Pearsons chi-square test to allow deciding on the inclusion of species. Data were exported from the Excel file to an Access file in which the number of strains having an MIC for a certain antibiotic were calculated. These data were set in a table that was subsequently exported to an Excel file. In this file breakpoints based on the EUCAST ECOFFs were indicated. The number of resistant strains was counted and resistance percentages were calculated. Exact confidence intervals for the binomial distribution were calculated using a visual basic application in Excel. A 95% symmetrical two-sided confidence interval was used with p= The lower and upper bound of confidence interval for the population proportion was calculated. Based on the Pearsons chi-square test, and where appropriate the Fischer exact test, significance of the differences were calculated. As for the differences between years, the chi square test has been used. It should be noted that differences seen here are not an indication of a trend. Trend cannot be calculated yet, since this needs 3 measurement points. Multi-resistance was determined by transforming the MIC data into resistant (R) and susceptible (S) using ECOFF breakpoints as provided by EUCAST. Number of antimicrobials to which a strain was resistant to was counted and cumulative percentages were calculated. The modal number of antimicrobials to which 50% of the strains was resistant was calculated. Graphical representations were prepared. 3 Results Results are shown in tables 2 to 51 and figures 1 to 32. The results are split up into the different animal species and different bacterial species. The division per bacterial species is because normal susceptibility of each may differ. Analysis per animal species allows determining differences between the animal species. Data are discussed only if a sufficient number of strains was obtained. 3.1 Poultry A total of 376 enterococci from poultry were tested for susceptibility. One hundred forty nine were E. faecalis, 63 E. faecium, 51 E. hirae and 14 were E. durans. Compared to 2011, there were quite more E. faecalis (2011: 81) and E. faecium (2011: 33) strains isolated. There were less E. hirae (2011:48) and only 3/75

4 few E. durans (2011:81) isolated. The adapted isolation method has favoured the isolation of E. faecium and E. faecalis but the inverse is true for the other two species tested. Nevertheless, sample size is not sufficient for attaining 170 strains of E. faecium and E. faecalis. Seen the low numbers of E. hirae and especially for E. durans, both from the E. faecium species group, and as one can see with the same normal susceptibility distribution for the antibiotics tested, it is no use to continue testing these species. In E. faecalis resistance was seen against all antibiotics except florfenicol. Resistance was mainly seen against tetracycline, erythromycin and streptomycin with at least half of the strains being resistant and even more than 85% of the strains being resistant to tetracycline. Resistance against the other antibiotics was less that 15%, with nearly 15% of the strains being resistant to salinomycin. Four strains were resistant to vancomycin. Resistance against this antibiotic shows cross-resistance to avoparcin, an antibiotic that was formerly used as growth promoter. Another antibiotic group formerly used as growth promoter and still in use as a coccidiostat, are the ionophores of which is salinomycin is tested here, against this antibiotic resistance was as high as 14,1%. Resistance against linezolid, a last resort antibiotic in the treatment of human infections with gram-positive bacteria was found in four strains. In E. faecium, no resistance against florfenicol, linezolid and vancomycin was seen. As for E. faecalis, resistance against tetracycline, erythromycin and streptomycin was high. Next to that high resistance was also found against quinupristin/dalfopristin, with 91,4% of the strains being resistant. Notable high resistances (nearly 40%) were noted against ampicillin and salinomycin. Resistance in E. hirae and E. durans is similar to what was found in E. faecium. These species are belonging to the same species group. One notable exception is the significantly lower resistance against ampicillin in E. hirae. Also here, resistance against salinomycin is attaining 40%. As a general, one can state that for the antibiotics against which resistance is high (erythromycin, tetracycline and streptomycin), it is so for the 4 species tested. As for the differences between the species, in general, prevalence of resistance in E. faecalis is significantly lower compared to E. faecium. Resistance prevalence in E. faecium did not differ with E. hirae and E. durans, though this may be due to the low number of strains tested. Resistance against salinomycin is also significantly lower in E. faecalis compared to E. faecium and E. hirae, but not with E. durans, most probably due to the low numbers of E durans tested. A more marked difference was seen for quinupristin/dalfopristin, with lower resistance for E. faecalis and high for the species of the E. faecium group. However, it should be noted that the normal MICs of this antibiotic for E. faecalis are substantially higher than for the species of the E. faecium group. This might have caused the differences seen. Resistance against the clinically important antibiotics linezolid and vancomycin was only seen in E. faecalis, however at low numbers. Only 10% of the E. faecalis strains were fully susceptible, and for the species of the E. faecium group, this was below 5%. In general strains were resistant to two to four antibiotics, which is also reflected by the high percentages of resistance against tetracycline, erythromycin or streptomycin. E. faecium was clearly most multi-resistant, with approximately 50% of the strains being resistant to 4 different antibiotics. One E. 4/75

5 faecalis strain was resistant up to 8 antibiotics and one E. faecium strain up to 7 different antibiotics. The E. faecalis strain remained susceptible to chloramphenicol, gentamicin and streptomycin and was resistant to ampicillin and vancomycin two important antibiotics in the treatment of enterococcal infections. The vancomycin resistant strains were the most resistant strains with the 4 strains being resistant to 8 or 7 different antibiotics. Most remained susceptible to ampicillin and gentamicin. None of the ampicillin resistant strains was resistant to gentamicin. As for the gentamicin resistant E. faecalis strains (4), all were also resistant to tetracycline but remained susceptible to ampicillin. One strain was also resistant to ciprofloxacin. Resistance against chloramphenicol, an antibiotic not used anymore is low, with a only 7 strains being resistant. The four linezolid resistant E. faecalis strains were co-resistant to 7 or 8 antibiotics, including vancomycin. Except one, all were susceptible to ampicillin and one was resistant to gentamicin. Striking also is that these strains were all resistant to salinomycin. 3.2 Pigs A total of 243 strains from pigs were tested. Twenty two were E. faecalis, 121 E. faecium, 85 E. hirae and 15 E. durans. Though these numbers are substantially higher than last year, few E. faecalis were isolated. The number of E. faecium has increased most but also the number of E. hirae has doubled. The number of E. durans remained stable. Seen the low number of isolates of the latter, it is of no use to include this species any longer. The number of strains from the E. faecium group outnumbered the E. faecalis strains largely. Seen the improved isolation method, the sample size should be increased to obtain sufficient E. faecalis strains. In E. faecalis from pigs, most resistance was seen against erythromycin and tetracycline, and then followed by streptomycin, gentamicin and chloramphenicol. The high prevalence of chloramphenicol resistance should be interpreted with care seen the low number of isolates included and the large confidence intervals. Other resistances were rare or absent, but also here, one should take into account the low number of isolates obtained and likewise the low sensitivity to find any resistant strain. In E. faecium, the number of strains tested is substantially higher compared to E. faecalis and the resistance prevalence is estimated more accurately. Here we see that against all antibiotics tested, there is resistance present. Highest resistance is present against quinupristin/dalfopristin. Half of the strains is resistant to tetracyclines. Against the other antibiotics, resistance is lower than a quarter of the strains, with for most antibiotics lower than 5%. Resistance against the clinically important antibiotics ampicillin is more than 17%. Gentamicin and vancomycin resistance remains low. Also in E. hirae, resistance was seen against all antibiotics. Seen the relatively higher number of strains isolated it is possible to compare the results from E. faecium and E. hirae, both from the same species group. There were no significant differences in resistance prevalence between E. faecium and E. hirae. As 5/75

6 such, the results from E. faecium and E. hirae can be superposed. The number of E. durans strains was too low to draw any conclusions but the levels of resistance were similar as those for the other species of the E. faecium group. In E. hirae, no one strain was fully susceptible, while in E. faecium, there were still 7 strains (approx. 6%) and in E. faecalis 3 strain (approx. 13%) susceptible. The insusceptibility of the species of the E. faecium group is mainly caused by the fact that most strains were resistant to quinupristin/dalfopristin. While multiresistance was very evident in E. faecium, with one strain resistant to as much as 10 antibiotics, most strains E. faecalis strains were resistant to maximal one antibiotic as shown by the fact that nearly 50% of the strains was resistant to 0/1 antibiotic. In E. faecalis, there was only on strain resistant to a maximum of 5 antibiotics, however, here, only 22 strains were tested. Also for E. hirae, there was one strain resistant to as much as 10 antibiotics, and approximately one third of the strains was susceptible or resistant to only one antibiotic. The highly multi-resistant E. faecium strains (resistant to more than eight antibiotics) were all resistant to ampicillin and vancomyin, two of the most important antibiotics in the treatment of enterococcal infections. They remained susceptible to gentamicin, but were resistant to linezolid. Vancomycin was typically associated with multi resistance, with only one strain being resistant to 5 antibiotics and the others being resistant against 8 to 10 antibiotics. This means that this resistance is coselected by quite some other antibiotics. It has been demonstrated that the usage of macrolides is associated with the maintenance of vancomycin resistance in enterococci. Similarly, linezolid resistance was associated with multi resistance, while ampicillin resistant strains could be susceptible to all other antibiotics. Also in E. hirae, vancomycin was typically associated with multi-resistance, including macrolide resistance. Two of the three strains were also ampicillin resistance, and all were linezolid resistance. These three strains remained susceptible to gentamicin. Ampicillin resistance was associated with resistance to at least 2 other antibiotics but never with gentamicin. 3.3 Veal calves While last year very few strains were obtained from veal calves, not allowing any analysis or conclusions, this year, 185 strains were obtained. Fifty eight were E. faecalis, 100 E. faecium, 17 E. hirae and 10 E. durans. While last year, the most isolated species was E. hirae (42 strains), this year, it was E. faecium. Also in bovines, E. durans is the lowest, and will be omitted for further inclusion. Seen the raise in E. faecium strains, it is not necessary anymore to include also the E. hirae. The number of samples should be increased to attain sufficient E. faecalis strains. In E. faecalis, the only antibiotic for which no resistance was noticed was salinomycin. Similarly to poultry and pigs, resistance against tetracycline (89,7%), erythromycin (82,8%) and streptomycin (74,1%) is highest. Half of the strains was resistant to chloramphenicol wich is extremely high for an antibiotic not used anymore since almost 20 years. Against the other antibiotics, little resistance is noted. 6/75

7 In E. faecium, no resistance was seen against linezolid. Similarly, high resistances were seen against tetracycline, erythromycin, and streptomycin, though significantly lower than for E. faecalis. Highest resistance was seen against quinupristin/dalfopristin, with 82% of the strains being resistant. Similar results were noted for E. hirae, but these strains were in general a bit more resistant than the E. faecium strain, though in most cases not significant, most probably due to the fact that few E. hirae strains were isolated. Though much less isolates were tested for E. durans, a similar resistance profile was found. Surprisingly in the tree most prevalent species, resistance against vancomycin has been detected. Typically also was that most strains the species of the E. faecium group were resistant to quinupristin/dalfopristin. In E. faecalis, approximately 10% of the strains was susceptible to all antibiotics tested. Fifty% of the strains were resistant to tree or more antibiotics. One strain was resistant to as much as 7 antibiotics. This strain was resistant to ampicillin, which is an important antibiotic in the treatment of E. faecalis infections. The strain remained however susceptible to vancomycin and gentamicin, but was ciprofloxacin resistant. The vancomycin resistant strain was resistant to 5 different antibiotics, typically also including erythromycin. In E. faecium, 7% of the strains remained susceptible while approximately 50% were resistant to 2 or more antibiotics, which is lower than for E. faecalis. This means that next to quinupristin/dalfopristin resistance, 50 % of the strains were also resistant to another antibiotic. One strain was resistant to 6, one to 7 and one up to 8 antibiotics. The strains resistant to 6 and 8 antibiotics were resistant to ampicillin but remained susceptible to gentamicin. The strain resistant to 7 antibiotics was susceptible to ampicillin and resistant to gentamicin. These 3 strains remained susceptible to vancomycin. The strain resistant to vancomycin was resistant to four antibiotics, typically including erythromycin resistance. Nearly 12% of the E. hirae strains remained susceptible and approximately half was resistant to one or more antibiotics. One strain was resistant up to 8 antibiotics. This strain was resistant to ampicillin but remained susceptible to gentamicin and vancomycin. The vancomycin resistant strain was resistant to as much as 5 antibiotics including erythromycin and linezolid. Though only 10 E. durans strains were included, half of the strains was resistant to 3 or more antibiotics. Two strains were resistant to 5 antibiotics. The 3 ampicilin resistant strains were resistant to 3 to 5 antibiotics and remained susceptible to gentamicin and vancomycin. 3.4 Bovines Contrary to isolates obtained from other species, most strains were E. hirae (61 strains) followed by E. faecium (58 strains), and E. faecalis (28 strains). As for the other animal species sampled, E.durans had the lowest isolation success with only 15 strains isolated. This was however relatively high compared to what is seen in other animal species but still the lowest isolation rates were from bovines. Also here it is of no use to continue testing E. durans. It is unclear what would be the best choice for the other species of the E. faecium group to be included since the isolation success for the two species were similar. Anyhow, seen the 7/75

8 low isolation success for E. faecalis, more samples need to be taken, and as such, more E. faecium strains will be isolated. Resistance in E. faecalis was highest for streptomycin, tetracycline and erythromycin. There was no resistance found against florfenicol, linezolid, salinomycin, quinupristin/dalfopristin and vancomycin. Resistance to chloramphenicol was almost 18% and resistance against the clinically important ampicillin and gentamicin was a bit more than 7%. Resistance in E. faecium was mainly seen against the antibiotic quinupristin/dalfopristin. Other resistances were lower than 30%, but the highest resistance percentages were also against tetracycline, erythromycin and streptomycin. One strain was resistant to vancomycine and 4 to ampicillin. Gentamicin resistance was absent and this was the sole antibiotic against which there was no resistance was found. Resistances against the other antibiotics were lower than 5%. Resistance of E. hirae against antibiotics was similar to what was found for E. faecium, with somehow a bit more resistance to ampicillin and vancomycin. Here no resistance was found against florfenicol. As for E. durans, few strains were isolated but the same trend in resistance as for the other species of the E. faecium species group was seen. Approximately 50% of the E. faecalis strains were resistant to two and more antibiotics. Nearly 30% of the strains remained fully susceptible. One strain was resistant to 5 antibiotics. This included ampicillin and gentamicin, rendering infections with such a strain difficult to treat. The strain remained susceptible to vancomycin, so treatment with this antibiotic is still possible. The other ampicillin resistant strain remained susceptible to gentamicin. In E. faecium, where more strains were tested, only 10% of the strain remained fully susceptible, but most strains were resistant to only one antibiotic (approx. 60%). Contrary, one strain was resistant to as much as 10 antibiotics, which makes it one of the most resistant strains isolated during this years surveillance. This strain remained only susceptible to ciprofloxacin and gentamicin. Only 20% of the strains were resistant to more than 3 antibiotics. All of the four ampicillin resistant strains remained susceptible to gentamicin and one was co-resistant to vancomycin. 3.5 Comparison between animal species Striking is the similarity that for each origin (animal species) and for each enterococcal species tested, the highest resistances were against tetracycline erythromycin and streptomycin. For the species in the E. faecium group, also quinupristin/dalfopristin should be taken into account. In poultry there is in general significantly more resistance against these antibiotics compared to the other animal species, though this is only visible when enough strains were tested and as such the confidence intervals are more tight, allowing to detect differences. 8/75

9 Resistance to chloramphenicol, an antibiotic not being used anymore for nearly 20 years is still present and especially in E. faecalis, and to a much lesser extend in E. faecium. This was especially true for strains from veal calves. Chloramphenicol resistance can be mediated by a gene encoding a chloramphenicol acetyl transferase, which does not give cross resistance with florfeniol. At the other hand there is the fex gene that has recently been described in Staphylococcus spp. but not yet in enterococci. Since florfenicol has been used mainly in ruminants, it may be that this resistance gene has been introduced in E. faecalis from bovines and is spreading. This resistance has also significantly increased compared to Further research is necessary to confirm this. Ampicillin resistance is mainly associated with E. faecium and this mainly in poultry, flowed by pigs. This type of resistance is chromosomally mediated, and a large part of its spread might be clonal. Vancomycin resistance has been detected in all animal species tested, however, not always in the same bacterial species. Percentage of resistant strains remains however low. 3.6 Comparison between 2011 and 2012 When comparing the data between 2011 and 2012, one needs to be cautious since, it may be a natural variation and not presenting a real trend in increasing or lowering of resistance and second, for some data, the number of strain tested were quite low. For some data, numbers were so low that no comparison could be made. Further surveillance will be enabling us to detect true trends. Statistical differences were seen on only tw occasion. For E. hirae and poultry and pigs, where there was a decrease in resistance for erythromycin and tetracycline. Care has to be taken not to over interpret the differences. 4 Conclusions This is the second large nationwide study on enterococci in Belgium. The number of strains included this year was substantially higher than before but still not ideal for most of the samples. Nevertheless it is clear that it is not useful to test E. durans anymore since on both occasions, few strains were detected. Since their morphological resemblance on Slantez and Bartley agar plates with the other species from the E. faecium group, they will be isolated and identified anyhow. As for E. hirae, isolation rates were much more favourable, however, lower than for E. faecium. Last year this was not the case. The inclusion of the NaCl supplemented broth in the isolation procedure seems to favour the isolation of E. faecium, rather than E. hirae. Therefor also this species will not be included anymore in the susceptibility tests, also because the resistance percentages were nearly similar to what is found in E. faecium. They were still included in this year s surveillance seen the outcome of the new method was uncertain and it was necessary to have enough data for detecting trends. It is clear that the behaviour of the species of the E. faecium group is similar and data may be compiled, strengthening trend analysis. Marked differences were seen between resistance in E. faecalis and the species from the E. faecium group (E. faecium, E. hirae and E. durans) concerning resistance to quinupristin/dalfopristin, a streptogramin antibiotic. Normal MICs of E. faecalis to this antibiotic is much higher compared to the species of the E. 9/75

10 faecium group. Moreover, there is partial cross-resistance of this antibiotic with the antibiotics of the macrolide-lincosamide-streptogramin (MLS) group of antibiotics. This may explain the high levels of resistance seen in the E. faecium group of bacteria, since most strains were resistant to erythromycin, a macrolide antibiotic. Erythromycin resistance is caused mostly by the presence of erm genes, encoding a methylase of the ribosomal RNA. Since macrolides and streptogramins have overlapping binding sites, this affects the susceptibility of the bacterium. Streptogramins however are always composed of a streptogramin A and a streptogramin B compound. The cross-resistance affects the binding of the B compound to the RNA, while the A compound remains active, resulting in a discrete increase of MICs, as seen in most of the strains in this surveillance. There exist however also specific resistance genes for streptogramins causing a high level of resistance. These high MICs are however more rarely encountered. A molecular investigation towards the genetic background of the resistance is the only way to determine the difference ( breakpoint ) between the high and low level resistances. Concerning multi-resistance, the species of the E. faecium group were more multi-resistant, though this is only on the account of quinupristin/dalfopristin resistance. In general 50% of the strains were resistant to 3-4 antibiotics. Never the less, E. faecalis could be resistant to as much as 8 antibiotics (poultry) and E. faecium to 10 antibiotics (pigs, bovines). This high multi- resistance represents however only a small fraction of the strains. Comparison between the two years should be interpreted with care and few significant differences were noted. When comparing the obtained results with those from The Netherlands (Maran 2012, data on strains isolated in 2011) similar results can be found. Also here most resistance was found against tetracycline, erythromycin and streptomycin, and for E. faecium also quinupristin/dalfoprisin. While no ampicillin resistance was found in E. faecalis in the Netherlands, we detected in Belgium. Levels of ampicillin resistance in E. faecium were similar. 10/75

11 Table 1. List of abbreviations Abbreviation AMP CHL CIP ERY FFN GEN LIN SAL STr SYN TET VAN Ampicillin Chloramphenicol Ciprofloxacin Erythromycin Florfenicol Gentamicin Linezolid Salinomycin Streptomycin Synercid (quinupristin/dalfopristin) Tetracycline Vancomycin 11/75

12 Table 2. Antibiotic resistance in commensal Enterococci from poultry. AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN E. faecalis N NR %R 6,7 2,7 2,7 72, ,7 14,1 51 2,7 86,6 2,7 CI 3,3-12 0,7-7 0,7-7 64, ,5-9 0,7-7 8, ,7-59 0, ,7-7 E. faecium N NR %R 38,9 1,2 8 74,1 0 1,9 0 37, ,4 78,4 0 CI 31,3-47 0,-4 4, , , , , , E. hirae N NR %R , ,3 21,6 92, CI , , , , , E. durans N NR %R 14, , ,9 42, ,9 0 CI 1, , , , , , /75

13 Tabel 3. Antibiotic resistance in commensal Enterococci from pigs. AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN E. faecalis N NR %R 0 18,2 4,5 63,6 0 18, ,8 0 81,8 0 CI ,2-40 0, , , , , E. faecium N NR %R 17,4 1,7 3,3 27,3 1,7 1,7 3,3 5 26,4 90,1 49,6 4,1 CI 11,1-25 0,2-6 0,9-8 19,6-36 0,2-6 0,2-6 0,9-8 1, , , ,4-59 1,4-9 E. hirae N NR %R 10,6 2,4 1,2 30,6 2,4 1,2 3,5 3,5 27,1 96,5 65,9 3,5 CI , , ,7-10 0, ,8-76 0,7-10 E. durans N NR %R 6, , ,3 0 CI 0, ,2-32 4, , , , , /75

14 Table 4 Antibiotic resistance in commensal Enterococci from veal calves. AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN E. faecalis N NR %R 1,7 51,7 5,2 82,8 1,7 6,9 1,7 0,0 74,1 1,7 89,7 1,7 CI ,2-55 1, , , , E. faecium N NR %R CI 4, ,2-7 32,2-52 0, ,6-9 27, , , E. hirae N NR %R 5,9 11,8 5,9 41,2 5,9 0 5,9 5,9 29,4 76,5 41,2 5,9 CI 0,1-29 1,5-36 0, ,4-67 0, ,1-29 0, , , ,4-67 0,1-29 E. durans N NR %R CI 6,7-65 0, , , , , /75

15 Table 5. Antibiotic resistance in commensal Enterococci from bovines. AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN E. faecalis N NR %R 7,1 17,9 0 46,4 0 7, ,7 0 57,1 0 CI 0,9-24 6, , , , , E. faecium N NR %R 6,9 1,7 3,4 24,1 1,7 0,0 1,7 3,4 19,0 82,8 25,9 1,7 CI 1, , , ,4-12 9, , , E. hirae N NR %R 8,2 1,6 1,6 32,8 0 1,6 4,9 9,8 24,6 72,1 39,3 8,2 CI 2, , jan/14 3, ,5-37 5, ,1-53 2,7-18 E. durans N NR %R 6,7 0 6, , ,3 33,3 0 CI 0, , , , , /75

16 Table 6. Antimicrobial resistance in in E. faecalis from different animal species. AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN Poultry N NR %R 6,7 2,7 2,7 72, ,7 14,1 51 2,7 86,6 2,7 CI 3,3-12 0,7-7 0,7-7 64, ,5-9 0,7-7 8, ,7-59 0, ,7-7 Pig N NR %R 0 18,2 4,5 63,6 0 18, ,8 0 81,8 0 CI ,2-40 0, , , , , veal calves N NR %R 1,7 51,7 5,2 82,8 1,7 6,9 1,7 0,0 74,1 1,7 89,7 1,7 CI ,2-55 1, , , , bovines N NR %R 7,1 17,9 0 46,4 0 7, ,7 0 57,1 0 CI 0,9-24 6, , , , , /75

17 Table 7. Antimicrobial resistance in in E. faecium from different animal species. AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN Poultry N NR %R 38,9 1,2 8 74,1 0 1,9 0 37, ,4 78,4 0 CI 31,3-47 0,-4 4, , , , , , Pig N NR %R 17,4 1,7 3,3 27,3 1,7 1,7 3,3 5 26,4 90,1 49,6 4,1 CI 11,1-25 0,2-6 0,9-8 19,6-36 0,2-6 0,2-6 0,9-8 1, , , ,4-59 1,4-9 Veal calves N NR %R CI 4, ,2-7 32,2-52 0, ,6-9 27, , , bovines N NR %R 6,9 1,7 3,4 24,1 1,7 0,0 1,7 3,4 19,0 82,8 25,9 1,7 CI 1, , , ,4-12 9, , , /75

18 Table 8. Antimicrobial resistance in in E. hirae from different animal species. AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN Poultry N NR %R , ,3 21,6 92, CI , , , , , Pigs N NR %R 10,6 2,4 1,2 30,6 2,4 1,2 3,5 3,5 27,1 96,5 65,9 3,5 CI , , ,7-10 0, ,8-76 0,7-10 veal calves N NR %R 5,9 11,8 5,9 41,2 5,9 0 5,9 5,9 29,4 76,5 41,2 5,9 CI 0,1-29 1,5-36 0, ,4-67 0, ,1-29 0, , , ,4-67 0,1-29 bovines N NR %R 8,2 1,6 1,6 32,8 0 1,6 4,9 9,8 24,6 72,1 39,3 8,2 CI 2, , jan/14 3, ,5-37 5, ,1-53 2, /75

19 Table 9. Antimicrobial resistance in in E.durans from different animal species. AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN Poultry N NR %R 14, , ,9 42, ,9 0 CI 1, , , , , , Pig N NR %R 6, , ,3 0 CI 0, ,2-32 4, , , , , Veal calves N NR %R CI 6,7-65 0, , , , , Bovines N NR %R 6,7 0 6, , ,3 33,3 0 CI 0, , , , , /75

20 Table 10 Antimicrobial resistance in E. faecalis from poultry Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 6,7 2,7 2,7 72,5 0,0 4,0 2,7 14,1 51,0 2,7 86,6 2,7 CI 3,3-12 0,7-7 0,7-7 64, ,5-9 0,7-7 8, ,7-59 0, ,7-7 20/75

21 Table 11 Antimicrobial resistance in E. faecium from poultry Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 38,9 1,2 8,0 74,1 0,0 1,9 0,0 37,7 63,0 91,4 78,4 0,0 CI 31,3-47 0,-4 4, , , , , , /75

22 Table 12 Antimicrobial resistance in E. hirae from poultry Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 2,0 2,0 0,0 39,2 0,0 0,0 0,0 37,3 21,6 92,2 51,0 0,0 CI , , , , , /75

23 Table 13 Antimicrobial resistance in E. durans from poultry Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 14,3 0,0 0,0 64,3 0,0 0,0 0,0 42,9 42,9 100,0 92,9 0,0 CI 1, , , , , , /75

24 Table 14 Antimicrobial resistance in E. faecalis from pigs Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 0,0 18,2 4,5 63,6 0,0 18,2 0,0 0,0 31,8 0,0 81,8 0,0 CI ,2-40 0, , , , , /75

25 Table 15. Antimicrobial resistance in E. faecium from pigs Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 17,4 1,7 3,3 27,3 1,7 1,7 3,3 5,0 26,4 90,1 49,6 4,1 CI 11,1-25 0,2-6 0,9-8 19,6-36 0,2-6 0,2-6 0,9-8 1, , , ,4-59 1,4-9 25/75

26 Table 16. Antimicrobial resistance in E. hirae from pigs Concetration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 10,6 2,4 1,2 30,6 2,4 1,2 3,5 3,5 27,1 96,5 65,9 3,5 CI , , ,7-10 0, ,8-76 0, /75

27 Table 17. Antimicrobial resistance in E. durans from pigs Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 6,7 0,0 0,0 26,7 0,0 0,0 0,0 0,0 20,0 80,0 33,3 0,0 CI 0, ,2-32 4, , , , , /75

28 Table 18. Antimicrobial resistance in E. faecalis from veal calves. Concentratie AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 1,7 51,7 5,2 82,8 1,7 6,9 1,7 0,0 74,1 1,7 89,7 1,7 CI ,2-55 1, , , , /75

29 Table 19. Antimicrobial resistance in E. faecium from veal calves. Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 9,0 1,0 2,0 42,0 2,0 1,0 0,0 3,0 37,0 82,0 47,0 1,0 CI 4, ,2-7 32,2-52 0, ,6-9 27, , , /75

30 Table 20. Antimicrobial resistance in E. hirae from veal calves. Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 5,9 11,8 5,9 41,2 5,9 0,0 5,9 5,9 29,4 76,5 41,2 5,9 CI 0,1-29 1,5-36 0, ,4-67 0, ,1-29 0, , , ,4-67 0, /75

31 Table 21. Antimicrobial resistance in E. durans from veal calves. Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 30,0 10,0 0,0 50,0 0,0 0,0 0,0 0,0 40,0 70,0 40,0 0,0 CI 6,7-65 0, , , , , /75

32 Table 22. Antimicrobial resistance in E. faecalis from bovines. Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 7,1 17,9 0,0 46,4 0,0 7,1 0,0 0,0 60,7 0,0 57,1 0,0 CI 0,9-24 6, , , , , /75

33 Table 23. Antimicrobial resistance in E. faecium from bovines. Cocentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 6,9 1,7 3,4 24,1 1,7 0,0 1,7 3,4 19,0 82,8 25,9 1,7 CI 1, , , ,4-12 9, , , /75

34 Table 24. Antimicrobial resistance in E. hirae from bovines. Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 8,2 1,6 1,6 32,8 0,0 1,6 4,9 9,8 24,6 72,1 39,3 8,2 CI 2, , , ,5-37 5, ,1-53 2, /75

35 Table 25. Antimicrobial resistance in E. durans from bovines. Concentration AMP CHL CIP ERY FFN GEN LZD SAL Str SYN TET VAN <= > N NR %R 6,7 0,0 6,7 20,0 0,0 6,7 0,0 0,0 20,0 73,3 33,3 0,0 CI 0, , , , , /75

36 Table 26. Multi-resistance in E. faecalis from poultry. Number of antimicrobials Number of strains % of bacteria Cumulative % ,7 10, ,1 24, ,8 43, ,9 84, ,1 94, ,0 96, ,7 97, ,0 99, ,7 100, ,0 100, ,0 100, ,0 100, ,0 100,0 36/75