In a global world, it is wholly understood that animals, humans, and environment are interconnected. The World Health Organisation (WHO), World Organisation for Animal Health (OIE), and Food and Agriculture Organisation (FAO), have joined efforts (8) to propose measures together against health risks at the animal-human-ecosystems interface. The OIE developed the “
The aim of this study was to detect the resistant phenotype profile of
Species of wild mammals included in the study
Order | Species | Scientific name | Origin | Scavenger | N | Isolates | % by sample – two samples per animal |
---|---|---|---|---|---|---|---|
Artiodactyla (n = 32) | Iberian ibex | CWFR-LA – Centre of Wild Fauna Recovery of La Alfranca (Spain); |
1 | 1 | 50.0 | ||
Mouflon | Hunting | 4 | 6 | 75.0 | |||
Red deer | Hunting | 9 | 6 | 33.3 | |||
Roe deer | Hunting | 1 | 3 | 150 | |||
Wild boar | Hunting | 17 | 22 | 64.7 | |||
Total | 32 | 38 | 59.4 | ||||
Carnivora (n = 19) | American mink | CWFR-LA | X | 6 | 8 | 66.6 | |
Badger | CWFR-LA | X | 3 | 5 | 83.3 | ||
Beech marten | CWFR-LA | X | 2 | 2 | 50.0 | ||
Common genet | CWFR-LA | X | 1 | 1 | 50.0 | ||
Common otter | CWFR-LA | X | 3 | 5 | 83.3 | ||
Red fox | CWFR-LA | X | 3 | 8 | 133.0 | ||
Weasel | CWFR-LA | X | 1 | 0 | 0 | ||
Total | 19 | 28 | 73.7 | ||||
Chiroptera (n = 1) | European free-tailed bat | CWFR-LA | 1 | 1 | 50.0 | ||
Erinaceomorpha (n =11) | Hedgehog | CWFR-LA | 11 | 15 | 68.2 | ||
Lagomorpha (n = 40) | Wild rabbit | Hunting | 38 | 27 | 35.5 | ||
Granada hare | Hunting | 2 | 1 | 100.0 | |||
Total | 40 | 28 | 35.0 | ||||
16 species | Total mammals | 103 | 111 | 53.9 |
There were 111 isolates recovered in which there were identified 15 different
A American mink; |
B Badger; |
Bm Beech marten; |
Cg Common genet; |
Co Common otter; |
G Granada hare; |
H Hedgehog; |
I Iberian ibex; |
M Mouflon; |
Ft European free-tailed bat; |
R Roe deer; |
Rd Red deer; |
Rf Red fox; |
W Weasel; |
Wb Wild boar; |
Wi Wild rabbit; |
Total n (%) | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 4 | 4 | 11 | 3 | 23 (20.7) | ||||||||||||
1 | 5 | 1 | 7 (6.3) | ||||||||||||||
2 | 2 (1.8) | ||||||||||||||||
1 | 1 (0.9) | ||||||||||||||||
1 | 1 | 1 | 2 | 1 | 11 | 17 (15.3) | |||||||||||
1 | 1 (0.9) | ||||||||||||||||
2 | 2 (1.8) | ||||||||||||||||
1 | 1 (0.9) | ||||||||||||||||
1 | 1 (0.9) | ||||||||||||||||
1 | 1 (0.9) | ||||||||||||||||
1 | 1 (0.9) | ||||||||||||||||
3 | 4 | 1 | 3 | 8 | 1 | 1 | 1 | 6 | 2 | 4 | 34 (30.6) | ||||||
2 | 2 (1.8) | ||||||||||||||||
3 | 2 | 1 | 1 | 7 (6.3) | |||||||||||||
1 | 1 | 1 | 8 | 11 (9.9) | |||||||||||||
Total staphylococci by mammal (n) | 8 | 5 | 2 | 1 | 5 | 1 | 15 | 1 | 6 | 1 | 3 | 6 | 8 | 0 | 22 | 27 | 111 |
Antibiotic phenotypical resistance to FA was high (64%), and in declining order resistance to other antibiotics was to PEN (42.3%), LIN (32.4%), FOX (20.7%), MUP (19.8%), and ERY (13.5%). The remaining studied antibiotics showed lower than 10% of isolates to be resistant, and the lower limit was SXT – 0% (Table 3). A low prevalence of MDR staphylococci isolates was detected in the studied population (7.2%; 8/111). The majority of FA-resistant isolates were CoNS (72.9%) (Table 4) obtained from rescued mammals (76.1%), where
Frequency of
Mammal species | N | PEN penicillin; |
FOX cefoxitin; |
FA fusidic acid; |
MUP mupirocin; |
ERY erythromycin; |
CLI clindamycin; |
LIN lincomycin; |
CIP ciprofloxacin; |
GEN gentamycin; |
TOB tobramycin; |
STR streptomycin; |
TE tetracycline; |
CL chloramphenicol; |
SXT sulfamethoxazole-trimethoprim; |
TEI teicoplanin |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
American mink | 8 | 4 | 1 | 6 | 1 | 3 | ||||||||||
Badger | 5 | 4 | 1 | 5 | 2 | 1 | ||||||||||
Beech marten | 2 | 1 | 1 | 1 | ||||||||||||
Granada hare | 1 | 1 | 1 | 1 | 1 | |||||||||||
Common genet | 1 | 1 | 1 | |||||||||||||
Common otter | 5 | 5 | 3 | 5 | 4 | |||||||||||
Hedgehog | 15 | 7 | 3 | 11 | 1 | 1 | 6 | 1 | 1 | |||||||
Mouflon | 6 | 1 | 2 | 1 | 3 | 1 | 1 | |||||||||
Free-tailed bat | 1 | 1 | 1 | |||||||||||||
Red deer | 8 | 1 | 2 | 1 | 3 | 2 | 1 | 3 | 1 | |||||||
Red fox | 7 | 3 | 3 | 5 | 1 | 2 | 4 | |||||||||
Roe deer | 2 | 2 | 2 | 1 | 1 | 1 | ||||||||||
Iberian ibex | 1 | 1 | 1 | 1 | 1 | |||||||||||
Weasel | 0 | |||||||||||||||
Wild boar | 22 | 2 | 1 | 11 | 7 | 3 | 1 | 4 | 4 | 3 | 1 | |||||
Wild rabbit | 27 | 14 | 7 | 20 | 9 | 4 | 2 | 10 | 3 | 1 | 1 | 2 | 3 | 1 | ||
Total | 111 | 47 | 23 | 71 | 22 | 15 | 7 | 36 | 3 | 2 | 1 | 10 | 13 | 2 | 0 | 1 |
% | 100 | 42.3 | 20.7 | 64 | 19.8 | 13.5 | 6.3 | 32.4 | 2.7 | 1.8 | 0.9 | 9.0 | 11.7 | 1.8 | 0 | 0.9 |
Results of the statistical analysis of
Antibiotics | Coagulase (N) | Resistant isolates, n (%) | Coagulase (N) | Resistant isolates, n (%) | Lower limit | Upper limit | |
---|---|---|---|---|---|---|---|
Fusidic acid | CoN coagulase-negative; |
62 (72.9) P ≤ 0.05; |
CoP coagulase-positive; |
9 (34.6) | 5.0918 | 1.9909 | 13.0225 |
Penicillin | CoN coagulase-negative; |
43 (50.6) P ≤ 0.05; |
CoP coagulase-positive; |
5 (19.2) | 4.3000 | 14.838 | 12.4609 |
Erythromyc in | CoN coagulase-negative; |
8 (9.4) P ≤ 0.05; |
CoP coagulase-positive; |
7 (26.9) | 3.5461 | 1.11434 | 11.0011 |
Origin (N) | Origin (N) | ||||||
Fusidic acid | CWFR-LA Centre of Wild Fauna Recovery of La Alfranca (Spain) |
35 (76.1) P ≤ 0.05; |
Hunting (65) | 36 (55.4) | 2.5631 | 5.9109 | 14.2860 |
Penicillin | CWFR-LA Centre of Wild Fauna Recovery of La Alfranca (Spain) |
27 (58.7) P ≤ 0.05; |
Hunting (65) | 20 (30.8) | 1.1114 | 3.1974 | 7.0353 |
Order (N) | Order (N) | ||||||
Fusidic acid | Artiodactyla vs (39) | 17 (43.59) | Carnivora P ≤ 0.05; |
22 (78.57) | 4.7461 | 11.249 | 1.0868 |
Lagomorpha P ≤ 0.05; |
21 (75.0) | 1.5755 | 8.2305 | 14.7275 | |||
Penicillin | Artiodactyla vs (39) | 7 (15.4) | Carnivora P ≤ 0.05; |
18 (64.3) | 14.2860 | 2.6702 | 5.27426 |
Erinaceomorpha P ≤ 0.05; |
7 (46.7) | 3.8820 | 25.3810 | 1.7476 | |||
Lagomorpha P ≤ 0.05; |
15 (53.6) | 1.3396 | 4.0000 | 15.9236 |
The probability of isolating PEN-resistant staphylococci was 4.3-fold higher for CoNS than for CoPS (Table 4). This probability was 8.2, 5.3 and 4.0-fold higher for Carnivora (64.3%; 18/28), Lagomorpha (53.6%; 15/28) and Erinaceomorpha (46.7%; 7/15), respectively, than for Artiodactyla (15.4%; 7/39). It was also 3.4 higher in scavenger mammals (64.3%; 22/28) than in non-scavenger animals (35%; 49/83). The prevalence of FOX-resistant isolates followed the same pattern as that observed for β-lactam antibiotics, without significant differences for CoPS and CoNS. Three
Although nasal samples are usually taken to isolate staphylococci from wild animals, since nasal secretions easily disseminate them by contact or proximity (14, 20, 21), we also isolated staphylococci from faeces, since we consider it an important route of dissemination of resistant bacteria into the environment. Staphylococci were mainly isolated from Carnivora (mostly scavengers) and Erinaceomorpha rescued by the CWFR-LA. Conversely, the order Lagomorpha gave the lower proportion of isolates, which was probably related to feeding habits, considering the percentages of detected in carnivores, invertebrate eaters, omnivores, and piscivores.
CoPS were mainly isolated from hunting mammals and the order Artiodactyla, including herbivorous and also omnivorous mammals which frequently visit human habitats where they could come into contact with human origin bacteria. It is of note that
Regarding antibiotic resistance, it should be mentioned that the five isolates from common otters (piscivores) were resistant to FA. It is important to highlight that the Ebro river was the habitat for four of these animals, because this circumstance reinforces the well-known importance of rivers for dissemination of antibiotics, mobile genetic elements of resistance and resistant bacteria (11, 16, 26). Since fusidic acid is only commercialised as a cream for staphylococcal infections, human sources could be associated with this FA resistance (3). Hunted mammals were the main source of staphylococci resistant to MUP and these predominated in omnivores and herbivores compared to carnivores. Therefore, resistance to this antibiotic appears to be related to the environment and plants, as other authors observed (11, 16, 26). Mupirocin is another topical antibiotic for prevention of human staphylococcal skin infections and eradication of MRSA. Consequently, resistance to MUP in wild animal isolates could also be related to human sources.
Beta-lactam antibiotics are the first-choice treatment for staphylococcal infections and a high proportion of isolates resistant to them (mainly CoNS) are frequently found in many human and animal environments (12), which can explain the high prevalence of resistance (42%) found in this study. Staphylococci which are non-susceptible to PEN were found to a greater extent in the Carnivora, Lagomorpha, and Erinaceomorpha orders than in Artiodactyla or animals with scavenger habits. Since penicillin was one of the most commonly sold antibiotics in Europe in 2013, 2014, and 2015 in both human and veterinary medicine (7), the source of this resistance could be linked to penicillin’s wide use on dairy, pig, and poultry farms, to its moderate bio-accumulation potential in plants (26), and to the exposure to dead animals from farms, this last source being especially relevant to carrion eaters (6). It is of note that we detected 19.8% of staphylococci resistant to PEN and FOX simultaneously to be methicillin resistant (MRS). MRSA has been widely studied and found in manure, farm-amended soils, and air pollution from livestock farms. Therefore, MRS could also be found in them and they could be a source of these resistant bacteria for wild mammals (12).
Resistance to TE was 12% in our study. This antibiotic is widely used in veterinary practice (7) and shows a moderate bio-accumulation potential in plants (26), which probably explains its extensive distribution in wild animals representing a large range of habits, environments, and dispersal over all the geographical areas studied. Despite the wide use of sulphonamides in humans and animals (mainly the combined SXT), particularly in urinary infections and bovine mastitis (11, 16), no isolates resistant to SXT were recovered in the study. Some unknown factors could act in the wild mammals studied to block their acquisition of staphylococci resistant to this antibiotic. We detected a low prevalence of staphylococci resistant to CIP (2.7%), in which three resistant isolates came from neonate or sub-yearling rabbits found in different geographical areas and different years, suggesting that these resistant isolates came from their family setting.
Isolates with ERY resistance were predominantly CoPS. This resistance has occasionally been associated with methicillin resistance in humans and farm animals (21), but only three isolates were also MRS in this study (2
We detected a low prevalence of MDR staphylococci isolates in the studied animals. The development of MDR is triggered by accumulation of resistance genes, vectored by plasmids or transposons that could be transferred between bacteria (19), and the antibiotic residues produced by humans and veterinary and agricultural activities. Antibiotic resistance is also produced without selective pressure, and this could explain the development of resistance in wildlife, in which the selective pressure is not so clear (2).
In conclusion, the majority of wild mammals included in this study came from geographical areas where agriculture and pig and sheep farms are widely distributed; however, the resistance values found in these wild mammals are lower than those to be expected if the use of antibiotics in farms had a direct influence on wildlife and its environment. On the other hand, resistance to antibiotics restricted to human use was widely disseminated in various wild animal species.