Abstract
Spread of multi-drug resistant (MDR) bacteria in natural environments pose a risk to human and animal health. Wild birds are considered to be reservoirs of human pathogens and vectors of antimicrobial resistance distribution in the environment. The aim of this study is to assess the occurrence of antibiotic resistant bacteria in isolates from bird specimens living in three agro-pastoral areas of the southeastern Sicily. We analyzed the microbiomes of the Eurasian Stone curlew Burhinus oedicnemus (Charadriiformes, Aves) and identified 91 Gram positive and 212 Gram negative strains, whose antimicrobial susceptibility to 11 and 9 antibiotic classes (respectively) was evaluated using agar disk diffusion test. Isolates showed significant levels of antimicrobial resistance, and a high percentage of MDR strains was found both between the Gram positive (49.4%) and the Gram negative (34.9%). Multi-drug resistance levels are higher among strains isolated in the beak and the eye than among enteric (faeces and cloaca) strains. Our results indicate high levels of MDR strains among wild bird populations, with a potential threat to wildlife and human populations.
Keywords: Microbiology, Environmental science, Environmental health, Epidemiology, Infectious disease, Bacteriology, Antibiotic resistant bacteria, Disk diffusion antimicrobial tests, Drug resistance, Gram negative bacteria, Gram positive bacteria, Burhinus oedicnemus, Sicily
Microbiology; Environmental science; Environmental health; Epidemiology; Infectious disease; Bacteriology; Antibiotic resistant bacteria; Disk diffusion antimicrobial tests; Drug resistance; Gram negative bacteria; Gram positive bacteria; Burhinus oedicnemus; Sicily
1. Introduction
The emergence of multiresistant bacteria in natural environments represents a potential hazard to human and animal health (Benskin et al., 2009; Allen et al., 2010; Radhouani et al., 2014). In recent years, substantial evidence has been provided linking high prevalence of antibiotic resistant bacterial strains in the environment with anthropogenic sources (Wellington et al., 2013; Atterby et al., 2016). The disproportionate administration of antibiotics to cattle and other livestock leads to their excretion into the environment in active forms (Berger et al., 1986; Gomes et al., 2017). The exposure of soil microorganisms to these molecules contributes to the development and amplification of resistance, with soil bacteria serving as reservoirs (Rysz and Alvarez, 2004). But effects on wildlife are less investigated, and the extent to which wild populations contribute to the spread of antibiotic resistance is still unknown. Given the increasing number of multidrug-resistant strains (resistant to three or more antibiotic classes), it is of crucial importance to understand the origins of antibiotic resistance in wild bird microbiome. Wild birds have rare contact with antimicrobial agents, thus they could serve as reservoir and potential spreaders of resistant bacteria (Guenther et al., 2010; Järhult et al., 2013; Bonnedahl and Järhult, 2014) and naturally evolving resistance genes (Janatova et al., 2014). This phenomenon is taking place on a global scale, and birds infected with multidrug-resistant bacteria can be found even in environments as remote from intensive farms as the Arctic (Benskin et al., 2009).
Several studies have evidenced the transmission of antibiotic resistant strains, belonging to the same clonal complex, between wildlife or livestock and humans. Conjugation and transfer of R plasmids occurs frequently in the environment, even between unrelated human and animal bacterial strains and in the absence of antibiotics (Kruse and Sorum, 1994). But our current knowledge of transmission routes is still insufficient, and more studies are necessary in order to evaluate the spread of antibiotic resistance genes (Arnold et al., 2016; Vittecoq et al., 2016).
Resistant bacteria of human and animal origin are likely to be transmitted to wild birds by contaminated food or water (Abulreesh et al., 2007; Bonnedahl et al., 2009; Guenther et al., 2010; Radhouani et al., 2012, 2014; Graham et al., 2014). Moreover, production of natural antibiotics by some bacteria and fungi (Keen and Montforts, 2012) occur naturally throughout the environment. The mixing of environmental bacteria with exogenous bacteria from direct deposition of faeces or urine, use of manure (Heuer et al., 2011) or of effluent flows (Wellington et al., 2013) provides the ideal selective and ecological conditions for new resistant strains to arise.
Many birds use faecal pellets as food resource, searching for invertebrates or worms and ingesting the antibiotics used for the livestock management as well. The few studies that identified potential sources of AMR and made comparisons across sites differing in contamination provide insights into the potential for wildlife to spread clinically relevant AMR. In order to clarify the role of the environment and of wildlife in the antibiotic resistance phenomenon emergence, more studies that analyze the complex ecology and the feedback between human and animal populations and the environment are needed.
Our study was conducted on the Eurasian Stone curlew Burhinus oedicnemus (Linnaeus, 1758), a palearctic species belonging to Charadriiformes that is of great conservation interest (Batten et al., 2013). B. oedicnemus is a Least Concern species in the Mediterranean and Macaronesian regions (BirdLife International, 2018), but in Italy its mating habitat is rapidly disappearing, and this bird is therefore classified as Vulnerable (Peronace et al., 2012; BirdLife International, 2018). This species nests on the ground, in open canopies characterized by sparse herbaceous vegetation (uncultivated drylands, grazed or halophilous pastures, river groves, etc.) and arboreal plantations (Hume and Kirwan, 2013). Farming practices have a major impact on B. oedicnemus' diet, as birds nesting in agro-pastoral areas mainly feed on invertebrates present in ruminant droppings (Spena et al., 2011).
Because of the strong relation between diet and microbiome composition (Brittingham et al., 1988), B. oedicnemus can be considered as an indicator of environmental contamination by multiresistant and potentially pathogenic bacteria deriving from human activities, especially farming.
The aim of this study is to assess the levels of antibiotic resistant bacteria in isolates from Eurasian Stone curlew nesting near human settlements in order to evaluate the spread of the antibiotic resistance in wild populations.
2. Materials and methods
2.1. Sampling sites
The samples were taken between July and August 2018 in a large area of the southeastern part of Sicily (Figure 1) in zones characterized by an agro-pastoral environment: the Gela Plain (Caltanissetta), the agroecosystems around Ragusa and the Thapsos Peninsula (Siracusa).
Figure 1.
Map showing the investigated areas in southern Sicily (Italy): a) Thapsos Peninsula (Siracusa), b) Gela Plain (Caltanissetta), and c) Ragusa. The coloured drops represent the sampled nests of the Burhinus oedicnemus. Photo credit of Burhinus oedicnemus from Thapsos Peninsula by MT Spena.
These areas host the most relevant nesting and wintering nuclei at regional level (Mascara and Sarà 2007; Tinarelli et al., 2008). The Gela Plain hosts the largest populations of the considered species and the number of pairs is estimated to be 150–200 (Tinarelli et al., 2008). Around Ragusa, the vegetation belongs to the alliance of Oleo sylvestris-Ceratonion siliquae Braun-Blanquet, and in the Thapsos Peninsula, carbonate bedrocks are covered by Thymbra capitata (L.) Cav. and subnitrophilous vegetation (Spena et al., 2011). In all of the three areas shown in Figure 1, cattle and, to a lesser extent, sheep and goats are present and their droppings represent a valuable food resource for the arthropod-rich fauna (Amat, 1986; Caccamo et al., 2011). The choice of study areas was also made on the basis of the consideration that in heavily populated areas, high background AMR levels often cloud observations while study sites with relatively low or well-defined AMR inputs, as in our case, enable us to quantify spatial patterns, pathways and processes that drive AMR dissemination (Arnold et al., 2016).
The individuals were captured from ground nests using a fall trap and cloacal, conjunctival and oral swabs for bacteriological survey were collected from each bird. Furthermore, whenever possible, a fresh faecal sample was taken. All birds were released immediately after sampling and returned to their nests. Our data indicate that nest disturbance was well tolerated by the birds. Indeed, all continued to incubate after being trapped (Spena et al., 2020).
2.2. Antimicrobial susceptibility testing
The samples from 61 Burhinus oedicnemus specimens were previously examined by bacteriological test for commensal and potentially pathogenic bacteria (Spena et al., 2020). The analysis had revealed the presence of 212 Gram negative and 91 Gram positive strains (Tables 1 and 2) (Spena et al., 2020).
Table 1.
List of tested Gram positive isolated from conjunctival swabs (C) and oral swabs (O).
| Bacterial Family | Bacterial species | Number of isolates |
||
|---|---|---|---|---|
| C | O | Total | ||
| Bacillaceae | Bacillus brevis | 1 | 1 | 2 |
| Bacillus cereus subsp mycoides | 1 | 1 | ||
| Bacillus fastidiosus | 1 | 1 | ||
| Bacillus licheniformis | 23 | 16 | 39 | |
| Bacillus megaterium | 5 | 5 | ||
| Bacillus pumilus | 1 | 1 | ||
| Bacillus spp | 1 | 1 | ||
| Bacillus subtilis | 2 | 2 | ||
| Exiguobacterium acetylicum | 1 | 1 | ||
| Enterococcaceae | Enterococcus faecalis | 1 | 3 | 4 |
| Enterococcus faecium | 2 | 2 | ||
| Lactobacillaceae | Lactobacillus rhamnosus | 1 | 1 | |
| Paenibacillaceae | Paenibacillus durus | 1 | 1 | |
| Staphylococcaceae | Staphylococcus aureus | 1 | 2 | 3 |
| Staphylococcus cohnii ssp cohnii | 1 | 1 | ||
| Staphylococcus epidermidis | 1 | 1 | ||
| Staphylococcus gallinarum | 1 | 1 | 2 | |
| Staphylococcus hominis | 2 | 1 | 3 | |
| Staphylococcus lentus | 1 | 1 | ||
| Staphylococcus saprophyticus | 1 | 1 | ||
| Staphylococcus sciuri | 4 | 11 | 15 | |
| Staphylococcus warneri | 1 | 1 | 2 | |
| Staphylococcus xylosus | 1 | 1 | ||
| Total | 46 | 45 | 91 | |
Table 2.
List of tested Gram negative isolated from fecal samples (F), cloacal swabs (Cl), conjunctival swabs (C) and oral swabs (O).
| Bacterial Family | Bacterial species | Number of isolates |
||||
|---|---|---|---|---|---|---|
| F | Cl | C | O | Total | ||
| Aeromonadaceae | Aeromonas sobria | 1 | 1 | |||
| Aeromonas hydrophila | 1 | 1 | ||||
| Enterobacteriaceae Group | Citrobacter amalonaticus | 5 | 4 | 9 | ||
| Citrobacter diversus | 4 | 3 | 7 | |||
| Citrobacter farmeri | 1 | 2 | 3 | |||
| Citrobacter freundii | 3 | 3 | 6 | |||
| Citrobacter spp | 13 | 18 | 1 | 32 | ||
| Enterobacter aerogenes | 2 | 6 | 1 | 9 | ||
| Enterobacter asburiae | 1 | 1 | 2 | |||
| Enterobacter cancerogenus | 2 | 1 | 2 | 5 | ||
| Enterobacter cloacae | 6 | 4 | 8 | 15 | 33 | |
| Enterobacter kobei | 3 | 2 | 5 | |||
| Enterobacter ludwigii | 2 | 2 | ||||
| Enterobacter spp | 1 | 1 | ||||
| Escherichia coli | 14 | 5 | 4 | 2 | 25 | |
| Escherichia hermannii | 1 | 1 | ||||
| Hafnia alvei | 6 | 2 | 3 | 2 | 13 | |
| Kluyvera ascorbata | 1 | 1 | ||||
| Leclercia adecarboxylata | 1 | 8 | 3 | 12 | ||
| Proteus mirabilis | 2 | 5 | 7 | |||
| Proteus vulgaris | 1 | 1 | 2 | |||
| Providencia rettgeri | 2 | 2 | ||||
| Salmonella enterica ssp enterica | 4 | 4 | ||||
| Serratia liquefaciens | 1 | 1 | ||||
| Serratia rubidaea | 1 | 1 | ||||
| Shigella dysenteriae | 1 | 1 | ||||
| Flavobacteriaceae | Chryseobacterium indologenes | 3 | 1 | 4 | ||
| Pseudomonadaceae | Pseudomonas aeruginosa | 2 | 3 | 1 | 6 | |
| Pseudomonas putida | 1 | 1 | ||||
| Pseudomonas stutzeri | 1 | 7 | 5 | 13 | ||
| Xanthomonadaceae | Stenotrophomonas maltophilia | 1 | 1 | |||
| Vibrionaceae | Vibrio mimicus | 1 | 1 | |||
| Total | 64 | 62 | 39 | 47 | 212 | |
We evaluated the antimicrobial susceptibility of all strains isolated (Supplementary data). The test was performed by the Disk diffusion method (Bauer et al., 1966) on Mueller-Hinton agar (Oxoid, Basingstoke, UK) in accordance to international standards (CLSI, 2017).
For Gram positive strains susceptibility to 20 antimicrobial agents belonging to 9 antibiotics classes was evaluated: Aminoglycosides [gentamicin (10 μg), tobramycin (10 μg)]; Carbapenems [imipenem (10 μg), meropenem (10 μg)]; Cephalosporins [ceftazidime (30 μg), cefovecin (10 μg), cefepime (30 μg), ceftaroline (30 μg)]; Fluoroquinolones [enrofloxacin (5 μg)]; .Glycopeptides [vancomycin (30 μg)]; Lincosamides [lincomycin (15 μg)]; Macrolides [erythromycin (15 μg)]; Penicillins [amoxicillin (30 μg), ampicillin (10 μg), oxacillin (1 μg), amoxicillin + clavulanic acid (30 μg), ampicillin + sulbactam (20 μg), ticarcillin + clavulanic acid (85 μg)]; Tetracyclines [minocycline (30 μg), tetracycline (30 μg)] (Liofilchem®, Teramo, IT).
For Gram negative strains susceptibility to 20 antimicrobial agents belonging to 11 antibiotics classes was evaluated: Aminoglycosides [gentamicin (10 μg), streptomycin (10 μg), tobramycin (10 μg)]; Carbapenems [imipenem (10 μg), meropenem (10 μg)]; Cephalosporins [cefotaxime (30 μg), cefotaxime + clavulanic acid (40 μg), ceftazidime (30 μg)]; Chloramphenicol [chloramphenicol (30 μg)]; Fluoroquinolones [ciprofloxacin (5 μg), enrofloxacin (5 μg)]; Monobactams [aztreonam (30 μg)]; Penicillins [amoxicillin (30 μg), amoxicillin + clavulanic acid (30 μg), ampicillin (10 μg)]; Polymyxins [colistin sulfate (10 μg)]; Quinolones [nalidixic acid (30 μg)]; Sulfonamides [co-trimoxazole (25 μg)]; Tetracyclines [minocycline (30 μg), tetracycline (30 μg)] (Liofilchem®, Teramo, IT).
The tests were interpreted by measuring the inhibition halo diameter of microorganism growth after incubation at 37 °C for 24 h. Isolates were considered either resistant or susceptible according to the manufacturer's instructions (Liofilchem®, 2019) based on CLSI guidelines (2017), in the case of Polymyxins according to Gales et al. (2001) and in the case of Lincosamides according to Rossney et al. (2007) (Tables 3 and 4). Isolates showing intermediate sensitivity were considered as resistant. Moreover, strains were considered multidrug resistant (MDR) when showing resistance to three or more antimicrobial classes (Magiorakos et al., 2012).
Table 3.
List of antibiotics used in Gram positive antimicrobial susceptibility tests and relative inhibition halos diameters for resistant (R), intermediate (I) and sensitive (S) strains.
| Class | Antibiotics | Code μg | Diameter (mm) |
||
|---|---|---|---|---|---|
| R < | I | S > | |||
| Aminoglycosides | gentamicin | CN 10 | 12 | 13–14 | 15 |
| tobramycin | TOB 10 | 12 | 13–14 | 15 | |
| Carbapenems | imipenem | IMI 10 | 16 | 17–21 | 22 |
| meropenem | MRP 10 | 16 | 17–21 | 22 | |
| Cephalosporins | ceftazidime | CAZ 30 | 14 | 15–17 | 18 |
| cefovecin | CVN 10 | 14 | 15–20 | 21 | |
| cefepime | FEP 30 | 14 | 15–17 | 18 | |
| ceftaroline | CPT 30 | 23 | 23 | ||
| Fluoroquinolones | enrofloxacin | ENR 5 | 17 | 18–20 | 21 |
| Glycopeptides | vancomycin | VA 30 | 14 | 15–16 | 17 |
| Lincosamides | lincomycin | MY 15 | 14 | 15–20 | 21 |
| Macrolides | erythromycin | E 15 | 13 | 14–22 | 23 |
| Penicillins | amoxicillin | AML 30 | 13 | 14–16 | 17 |
| amoxicillin + clavulanic acid | AUG 30 | 19 | 20 | ||
| ampicillin Staphylococcus spp. |
AMP 10 | 13 28 |
14–16 | 17 29 |
|
| ampicillin + sulbactam | AMS 20 | 11 | 12–14 | 15 | |
| oxacillin Staphylococcus spp. coagulase - |
OX 1 | 10 18 |
11–12 | 13 19 |
|
| ticarcillin + clavulanic acid | TIM 85 | 22 | 23 | ||
| Tetracyclines | minocycline | MN 30 | 14 | 15–18 | 19 |
| tetracycline | TE 30 | 11 | 12–14 | 15 | |
Table 4.
List of antibiotics used in Gram negative antimicrobial susceptibility tests and relative inhibition halos diameters for resistant (R), intermediate (I) and sensitive (S) strains.
| Class | Antibiotics | Code μg | Diameter (mm) |
||
|---|---|---|---|---|---|
| R < | I | S > | |||
| Aminoglycosides | gentamicin | CN 10 | 13 | 14–15 | 16 |
| streptomycin | S 10 | 11 | 12–14 | 15 | |
| tobramycin | TOB 10 | 13 | 14–15 | 16 | |
| Carbapenems | imipenem | IMI 10 | 19 | 20–22 | 23 |
| meropenem | MRP 10 | 19 | 20–22 | 23 | |
| Cephalosporins | cefotaxime | CTX 30 | 22 | 23–25 | 26 |
| cefotaxime + clavulanic acid | CTL 40 | 22 | 23–25 | 26 | |
| ceftazidime | CAZ 30 | 17 | 18–20 | 21 | |
| Chloramphenicol | chloramphenicol | C 30 | 12 | 13–17 | 18 |
| Fluoroquinolones | ciprofloxacin | CIP 5 | 19 | 20–21 | 22 |
| enrofloxacin | ENR 5 | 15 | 16–20 | 21 | |
| Monobactams | aztreonam | ATM 30 | 17 | 18–20 | 21 |
| Penicillins | amoxicillin | AML 30 | 13 | 14–17 | 18 |
| amoxicillin + clavulanic acid | AUG 30 | 13 | 14–17 | 18 | |
| ampicillin | AMP 10 | 13 | 14–16 | 17 | |
| Polymyxins | colistin sulfate | CS 10 | 11 | 12–16 | 17 |
| Quinolones | nalidixic acid | NA 30 | 13 | 14–18 | 19 |
| Sulfonamides | co-trimoxazole | SXT 25 | 13 | 14–15 | 16 |
| Tetracyclines | minocycline | MN 30 | 12 | 13–15 | 16 |
| tetracycline | TE 30 | 11 | 12–14 | 15 | |
Strains of the same bacterial species found in both faeces and cloacal samples and exhibiting the same resistance profile were considered to be equivalent and therefore only counted once.
2.3. Statistical analysis
As the genera Bacillus and Staphylococcus have been found to be the most frequently representative Gram positive in our samplings, and Enterobacter and Citrobacter the most representative among Gram negative, we focused our analysis of the percentage of resistance of the isolates to these strains. Using R-3.6.0, we performed a t-test to determine if a significant difference exists between the means of two groups of microbiomes examined (more specifically beak vs. eye for Bacillus, Staphylococcus and Enterobacter spp. and faeces vs. cloaca for Citrobacter and Enterobacter spp.). We fixed the significance limit for the statistical analyses at P = 0.05. In addition, we used a 2-sample Kolmogorov-Smirnov test to evaluate the difference in AMR levels distribution between different isolates, using the R-3.6.0 ks.test function.
Finally, we evaluated the extent to antimicrobial resistance levels are related to direct contact with environmental pathogens. In order to do this, we compared the resistance levels recorded in Gram negative strains likely originating from direct contact with the environment (isolated from eye and beak) with those recorded in enteric Gram negative strains isolated from faeces and cloaca using Fisher's Exact Test, fixing the significance limit at P = 0.05.
3. Results
The overall resistance to antibiotics of all Gram positive and Gram negative isolates points out a high resistance to some of tested molecules. The resistance patterns varied from one to eighteen in Gram positive and from one to twelve in Gram negative strains.
Gram positive strains often exhibited high values of resistance (Table 5), and multidrug resistance occurred in 45 strains (49.4%) (Supplementary data).
Table 5.
Numbers (n) and percentages (%) of Resistant Gram positive strains for single molecules (first couple of values) and their classes (second couple of values).
| Class | Antibiotics | n. Res | % Res | n. Res | % Res | |
|---|---|---|---|---|---|---|
| Aminoglycosides | gentamicin | 17 | 18.7 | 36/182 | 19.8 | |
| tobramycin | 19 | 20.9 | ||||
| Carbapenems | imipenem | 4 | 4.4 | 8/182 | 4.4 | |
| meropenem | 4 | 4.4 | ||||
| Cephalosporins | III generation | ceftazidime | 82 | 90.1 | 120/182 | 65.9 |
| cefovecin | 38 | 41.7 | ||||
| IV generation | cefepime | 35 | 38.5 | 35/91 | 38.5 | |
| V generation | ceftaroline | 15 | 16.5 | 15/91 | 16.5 | |
| Fluoroquinolones | enrofloxacin | 6 | 6.6 | 6/91 | 6.6 | |
| Glycopeptides | vancomycin | 17 | 18.7 | 17/91 | 18.7 | |
| Lincosamides | lincomycin | 32 | 35.2 | 32/91 | 35.2 | |
| Macrolides | erythromycin | 24 | 26.4 | 24/91 | 26.4 | |
| Penicillins | Aminopenicillins | amoxicillin | 25 | 27.5 | 47/182 | 25.8 |
| ampicillin | 22 | 24.2 | ||||
| Isoxacillin | oxacillin | 74 | 81.3 | 74/91 | 81.3 | |
| Penicillins + β-lactamase inhibitors | amoxicillin + clav ac | 7 | 7.7 | 16/273 | 5.9 | |
| ampicillin + sulbactam | 6 | 6.6 | ||||
| ticarcillin + clav ac | 3 | 3.3 | ||||
| Tetracyclines | minocycline | 2 | 2.2 | 4/182 | 2.2 | |
| tetracycline | 2 | 2.2 | ||||
The majority of the Gram positive isolates were resistant to ceftazidime (90.1%) and oxacillin (81.3%). Very low levels of resistance were found against Tetracyclines and Carbapenems (from respectively 2,2% to and 4.4%). Seven Gram positive strains (7.7%) were susceptible to all tested antibiotics. This analysis only included 207 of the 212 strains, as 5 strains of faecal origin presented an identical resistance profile to that of cloacal strains isolated from the same individual. They were therefore considered equivalent and only counted once.
Some Gram negative isolates displayed a wide antibiotic resistance (Table 6) and multidrug resistance occurred in 74 strains (35.7%) (Supplementary data).
Table 6.
Numbers (n) and percentages (%) of Resistant Gram negative strains for single molecules (first couple of values) and their classes (second couple of values).
| Class | Antibiotics | n. res | % res | n. res | % res | |
|---|---|---|---|---|---|---|
| Aminoglycosides | gentamicin | 13 | 6.3 | 107/621 | 17.2 | |
| streptomycin | 73 | 35.3 | ||||
| tobramycin | 21 | 10.1 | ||||
| Carbapenems | imipenem | 10 | 4.8 | 13/414 | 3.1 | |
| meropenem | 3 | 1.4 | ||||
| Cephalosporins | III generation | ceftazidime | 18 | 8.7 | 25/414 | 6 |
| cefotaxime | 7 | 3.4 | ||||
| Cephalosporins + β-lactamase inhibitors | cefotaxime + clav ac | 2 | 1 | 2/207 | 1 | |
| Fluoroquinolones | ciprofloxacin | - | - | 7/414 | 1.7 | |
| enrofloxacin | 7 | 3.4 | ||||
| Monobactams | aztreonam | 16 | 7.7 | 16/207 | 7.7 | |
| Penicillins | Aminopenicillins | amoxicillin | 109 | 52.7 | 186/414 | 44.9 |
| ampicillin | 77 | 37.2 | ||||
| Penicillins + β-lactamase inhibitors | amoxicillin + clav ac | 95 | 45.9 | 95/207 | 45.9 | |
| Phenicols | chloramphenicol | 7 | 3.4 | 7/207 | 3.4 | |
| Polymyxins | colistin sulfate | 147 | 71 | 147/207 | 71 | |
| Quinolones | nalidixic acid | 20 | 9.7 | 20/207 | 9.7 | |
| Sulfonamides | co-trimoxazole | 9 | 4.3 | 9/207 | 4.3 | |
| Tetracyclines | minocycline | 16 | 7.7 | 32/414 | 7.7 | |
| tetracycline | 16 | 7.7 | ||||
The majority of the Gram negative isolates were resistant to colistin sulfate (70.7%) and amoxicillin (52.3%). Low levels of resistance (3.1%) were found against Carbapenems also for the Gram negative isolates, but no resistance was found against Fluoroquinolones.
The most abundant MDR pattern observed in Gram negative was combined resistance to Aminoglycosides, Penicillins and Polymyxins while in Gram positive was combined resistance to Cephalosporins, Penicillins and Lincosamides.
Comparing the resistance percentages based on the sample, the highest values were found in the beak and eye isolates. Figure 2 shows the record distribution of the 177 strains according to the resistance to antibiotics of either the Gram positive or the Gram negative bacteria.
Figure 2.
Antibiotic resistance distribution of Gram positive (left, n = 91) and Gram negative (right, n = 86) isolates from beak and eye.
Gram negative strains isolated from the eye and the beak display considerably higher levels of resistance compared to enteric Gram negative (isolated from faeces and cloacal swabs). This difference is highly significant for a range of antibiotics, including gentamicin (P = 0.007), ceftazidime (P = 0.00001), aztreonam (P = 0.00009) and nalidixic acid (P = 0.0002) (Table 7).
Table 7.
Comparison of the resistance number recorded in Gram negative strains isolated from fecal samples (F) and cloacal swabs (Cl) (n. 121) vs. Gram negative strains isolated from conjunctival swabs (C) and oral swabs (O) (n. 86) by Fisher's Exact Test.
| Antibiotics | Number resistant strains |
P Fischer Values | |
|---|---|---|---|
| F + Cl | C + O | ||
| gentamicin | 3 (2.5) | 10 (11.6) | 0.008 |
| streptomycin | 37 (30.6) | 36 (41.8) | 0.08 |
| tobramycin | 8 (6.6) | 13 (15.1) | 0.06 |
| Imipenem | 4 (3.3) | 6 (7) | 0.32 |
| meropenem | 0 | 3 (3.5) | 0.07 |
| ceftazidime | 2 (1.7) | 16 (18.6) | 0.00001 |
| cefotaxime | 2 (1.7) | 5 (5.8) | 0.12 |
| cefotaxime + clav ac | 0 | 2 (2.3) | 0.16 |
| chloramphenicol | 5 (4.1) | 2 (2.3) | 0.70 |
| ciprofloxacin | - | - | - |
| enrofloxacin | 2 (1.7) | 5 (5.8) | 0.12 |
| Aztreonam | 2 (1.7) | 14 (16.3) | 0.00009 |
| Amoxicillin | 56 (46.3) | 53 (61.6) | 0.02 |
| Ampicillin | 41 (33.9) | 36 (41.9) | 0.19 |
| amoxicillin + clav ac | 50 (41.3) | 45 (52.3) | 0.09 |
| colistin sulfate | 80 (66.1) | 67 (77.9) | 0.03 |
| nalidixic acid | 4 (3.3) | 16 (18.6) | 0.0002 |
| co-trimoxazole | 3 (2.5) | 6 (7) | 0.16 |
| minocycline | 8 (6.6) | 8 (9.3) | 0.44 |
| tetracycline | 10 (8.3) | 6 (7) | 1.00 |
( ) = percentage.
Within-genera comparison suggests that this difference is caused by the different composition of the enteric and the beak/eye microbiomes. MDR levels also tends to be higher in bacteria found in the beak or eye when compared to enteric bacteria from the same genus, although such differences are not statistically significant (Table 8).
Table 8.
Number of MDR strains divided into bacterial genera and according to the source sample.
| Genera | F + Cl |
C + O |
P | ||
|---|---|---|---|---|---|
| n. MDR/n. total strains | % | n. MDR/n. total strains | % | ||
| Aeromonas spp. | 0/0 | - | 1/2 | 50 | - |
| Citrobacter spp. | 10/49 | 20.4 | 5/8 | 62.5 | 0.13 |
| Chryseobacterium spp. | 0/0 | - | 4/4 | 100 | - |
| Enterobacter spp. | 8/26 | 30.8 | 19/31 | 61.3 | 0.23 |
| Escherichia spp. | 4/19 | 21.1 | 1/6 | 16.7 | 1.00 |
| Hafnia spp. | 0/8 | 0.00 | 1/5 | 20 | 0.43 |
| Leclercia spp. | 0/1 | 0.00 | 3/11 | 27.3 | 1.00 |
| Proteus spp. | 4/7 | 57.1 | 1/2 | 50 | 1.00 |
| Providencia spp. | 1/2 | 50 | 0/0 | - | - |
| Pseudomonas spp. | 3/6 | 50 | 5/14 | 35.7 | 1.00 |
| Serratia spp. | 1/1 | 100 | 1/1 | 100 | 1.00 |
| Shigella spp. | 1/1 | 100 | 0/0 | - | - |
| Stenotrophomonas spp. | 0/0 | - | 1/1 | 100 | - |
Among the strains treated with antimicrobial classes common for Gram positive and Gram negative bacteria, beak microbiomes show a much higher tolerance against Aminoglycosides, Penicillins and Tetracyclines than eye microbiomes do. The resistance against Cephalosporins show opposite trends for Gram positive and Gram negative bacteria in beak and eye, as the Gram negative resistance is much higher in the beak than in the eye, in contrast to the Gram positive microbiomes where beak and eye bacteria show an almost equal resistance against Cephalosporins (Supplementary data).
The minimum levels of AMR found in Enterobacter spp. is 10% (Figure 3). A similar value is found among Staphylococcus spp. from eye isolates, with a slightly lower value found in beak isolates (Figure 3). Citrobacter spp. was recorded only four times in the beak and in the eye, whilst it has been recorded often in fecal and cloacal samples (Figure 3).
Figure 3.
Antibiotic resistance distribution of Bacillus and Staphylococcus (Gram positive) and Enterobacter (Gram negative) in beak and eye (a, upper panel) and Enterobacter and Citrobacter (Gram negative) in faeces and cloaca (b, lower panel).
Comparison between beak and eye isolates reveals no significant difference in AMR levels for several bacterial genera (Bacillus, t = 0.754, df = 50;Staphylococcus, t = 0.406, df = 28; Enterobacter, t = 1.572, df = 27) (beak and eye). Similarly, there is no significant difference in mean AMR levels between the most common genera of bacteria isolated from the faeces and from the cloaca (Enterobacter; t = 0.463, df = 24; Citrobacter, t = 0.966, df = 47). However, the spectrum of antibiotic resistance differs markedly between isolates (Figures 2 and 3). Kolmogorov-Smirnov (KS) test showed significant differences between the beak and the eye for Bacillus (P = 0.3645), Staphylococcus (P = 0.978) and Enterobacter, (P = 0.3504); and between faeces and cloaca for Enterobacter (P = 0.9998) and for Citrobacter (P = 0.7154) (a high p-value in the KS-test indicates a high likelihood that samples come from two distinct distributions).
4. Discussion
Our results evidence the role of wild birds as reservoirs and potential vectors of AMR bacteria. B. oedicnemus' feeding habits put it in close contact with bacteria from livestock, making it an excellent indicator of environmental contamination. To the best of our knowledge, B. oedicnemus is the only significant source of AMR dissemination identified in the areas were the study was conducted.
The range anti-microbial resistance levels in wild birds reported in the literature is broad. Such variations can often be attributed to differences in habitat (Carter et al., 2018), as demonstrated by several studies that measure resistance levels in isolates from birds with different lifestyles or captured in different environments (Shobrak and Abo-Amer, 2014; Atterby et al., 2016; Giacopello et al., 2016; Ramey et al., 2018; Tormoehlen et al., 2019). Our results are consistent with those reported in similar investigations, but strongly differ from other results found in the literature. Atterby et al. (2016) detected lower resistance levels than ours to all tested molecules in birds from a remote location in Southcentral Alaska, but found higher percentages for Cephalosporins, Quinolones and for Tetracyclines in a more urban setting, with similar levels of Aminoglycosid resistance. Shobrak and Abo-Amer (2014) report higher values in both migratory and non-migratory birds, but found no resistance to Aminoglycosid in non-migratory ones. We have, however, detected higher frequency of Polymyxins resistance compared to their values (24% in non-migratory and 37.5% in migratory birds). Botti et al. (2013) found lower percentage values than ours above all for Polymyxins in wildlife of Nord Italy.
Colistin sulfate is typically used as a last resort antibiotic to treat of MDR infections in humans, and has been regularly employed in veterinary medicine both as treatment and a preventative measure of Gram negative gastrointestinal infections. On 27 July 2016, the European Commission issued a Decision (EMA/CVMP/CHMP/231573/2016) which limits the use of colistin in the veterinary field and prohibits its use in combination with other antibiotics (European Commission, 2016). In the coming years, Italy will have to commit itself to reducing consumption within "desirable" values. In this regard, countries such as Italy and Spain, considered "high and moderate consumers", will have to set the goal of reducing consumption from 20-25 mg/population correction unit (currently recorded) to 5 mg/population correction unit of colistin (minimum target level).
Resistance levels against carbapenems and fluoroquinolone, whose use in veterinary medicine is restricted, are low compared to other classes of antibiotics. This supports the hypothesis that contact with livestock is the main cause of AMR spread in wild populations.
Both Gram positive as Gram negative strains display some resistance against Carbapenems. The resistance to a class of antibiotics generally used as an emergency measure against MDR bacteria together with the high resistance to other antibiotic classes, constitutes a potential threat to wildlife and therefore deserves further investigation.
Our results indicate a significant difference in the spectrum of antibiotic resistance between isolates from beak and eye and between faeces and cloaca. This suggests that an evaluation of the microbial flora aimed at obtaining epidemiological information must include sampling carried out on different regions of the body. In particular, our results show that a comprehensive evaluation of the intestinal microbiome must rely not only on cloacal swabs, but also on faecal samples.
5. Conclusion
Evaluation of antibiotic resistance levels in wildlife (especially in wild birds) is often limited to a few pathogenic bacteria such as Escherichia coli, Enterococcus spp., and Salmonella spp. As a consequence, little is known about the broader diversity of antibiotic resistant bacteria in wildlife and the environment (Carter et al., 2018). In our research, we examined 303 strains belonging to 22 genera (11 families) finding AMR in all of them. Many strains belonged to commensal bacterial species of the body sites from which they were isolated (eye, beak, intestine), suggesting the existence of more diffusion routes of resistant strains than the fecal deposition route typically reported in the literature (Guenther et al., 2010; Järhult et al., 2013; Shobrak and Abo-Amer, 2014). Our results show that commensal bacteria play an important role in the transmission of anti-microbial resistance, but further epidemiological studies are necessary in order to gain a more detailed understanding of this complex phenomenon.
Declarations
Author contribution statement
Maria Foti, Rosario Grasso: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Vittorio Fisichella, Antonietta Mascetti, Manuel Andrea Zafarana, Maria Grasso: Performed the experiments.
Marco Colnaghi: Analyzed and interpreted the data; Wrote the paper.
Maria Teresa Spena: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Competing interest statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
Acknowledgements
All the authors are grateful for the valuable field assistance of A. and C. Calvo, G. Debinja, D. Falzolgher, F. Giugno, F. Giunta, G. Occhipinti, D. Pepi, E. Russo and S. Tortorici. RG and MTS wish to express their sincere thanks to A. Sorace (Istruttoria), P. Genovesi (Gestione Patrimonio Faunistico Nazionale e mitigazione danni e impatti) and E. Morroni (ISPRA) for supporting the capture of Burhinus oedicnemus (Prot. 44648/T-A31), and G. Qualera (Assessorato Regionale dell'Agricoltura, dello Sviluppo rurale e della Pesca Mediterranea).
Appendix A. Supplementary data
The following is the supplementary data related to this article:
References
- Abulreesh H., Goulder R., Scott G.W. Wild birds and human pathogens in the context of ringing and migration. Ringing Migr. 2007;23:193–200. [Google Scholar]
- Allen H.K., Donato J., Wang H.H., Cloud-Hansen K.A., Davies J., Handelsman J. Call of the wild: antibiotic resistance genes in natural environments. Nat. Rev. Microbiol. 2010;8:251–259. doi: 10.1038/nrmicro2312. [DOI] [PubMed] [Google Scholar]
- Amat J.A. Information on the diet of the Stone curlew Burhinus oedicnemus in Doñana, southern Spain. Hous. Theor. Soc. 1986;33:71–73. [Google Scholar]
- Arnold K.E., Williams N.J., Bennett M. Disperse abroad in the land: the role of wildlife in the dissemination of antimicrobial resistance. Biol. Lett. 2016;12:20160137. doi: 10.1098/rsbl.2016.0137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Atterby C., Ramey A.M., Hall G.G., Järhult J., Börjesson S., Bonnedahl J. Increased prevalence of antibiotic-resistant E. coli in gulls sampled in Southcentral Alaska is associated with urban environments. Infect. Ecol. Epidemiol. 2016;6 doi: 10.3402/iee.v6.32334. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Batten L.A., Bibby C.J., Clement P., Elliott G.D., Porter R.F. Poyser; London: 2013. Red Data Birds in Britain. [Google Scholar]
- Bauer A.W., Kirby W.M.M.M., Sherris J.C., Turck M.A. Antibiotic susceptibility testing by a standardised single disc method. Am. J. Clin. Pathol. 1966;45:493–496. [PubMed] [Google Scholar]
- Benskin C.M., Wilson K., Jones K., Hartley I.R. Bacterial pathogens in wild birds: a review of the frequency and effects of infection. Biol. Rev. 2009;84:349–373. doi: 10.1111/j.1469-185X.2008.00076.x. [DOI] [PubMed] [Google Scholar]
- Berger K., Peterson B., Buening-Pfaune H. Persistence of drugs occurring in liquid manure in the food chain. Arch. Leb. 1986;37:99–102. [Google Scholar]
- BirdLife International . 2018. Burhinus oedicnemus. The IUCN Red List of Threatened Species 2018: e.T45111439A132038252. Downloaded on 18 July 2019. [Google Scholar]
- Bonnedahl J., Järhult J.D. Antibiotic resistance in wild birds. Ups. J. Med. Sci. 2014;119:113–116. doi: 10.3109/03009734.2014.905663. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bonnedahl J., Drobni M., Gauthier-Clerc M., Hernandez J., Granholm S., Kayser Y., Melhus A., Kahlmeter G., Waldenstrom J., Johansson A., Olsen B. Dissemination of Escherichia coli with CTX-M Type ESBL between humans and yellow-legged gulls in the South of France. PloS One. 2009;4 doi: 10.1371/journal.pone.0005958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Botti V., Navillod F.V., Domenis L., Orusa R., Pepe E., Robetto S., Guidetti C. Salmonella spp. and antibiotic-resistant strains in wild mammals and birds in north-western Italy from 2002 to 2010. Vet. Ital. 2013;49:195–202. [PubMed] [Google Scholar]
- Brittingham M.C., Temple S.A., Duncan R.M. A survey of the prevalence of selected bacteria in wild birds. J. Wildl. Dis. 1988;24:299–307. doi: 10.7589/0090-3558-24.2.299. [DOI] [PubMed] [Google Scholar]
- Caccamo C., Pollonara E., Baldaccini N.E., Giunchi D. Diurnal and nocturnal ranging behaviour of Stone curlews Burhinus oedicnemus nesting in river habitat. Ibis. 2011;153:707–720. [Google Scholar]
- Carter D.L., Docherty K.M., Gill S.A., Baker K., Teachout J., Vonhof M.J. Antibiotic resistant bacteria are widespread in songbirds across rural and urban environments. Sci. Total Environ. 2018;627:1234–1241. doi: 10.1016/j.scitotenv.2018.01.343. [DOI] [PubMed] [Google Scholar]
- Clinical Laboratory Standards Institute - CLSI . CLSI document M100S; Wayne, Pennsylvania, USA: 2017. Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Seventh Informational Supplement. [Google Scholar]
- European Commission . 27 July 2016. EMA/CVMP/CHMP/231573/2016 Updated Advice on the Use of Colistin Products in Animals within the European Union: Development of Resistance and Possible Impact on Human and Animal Health.http://www.ema.europa.eu/ema/index.jsp?curl=pages/news_and_events/news/2016/07/news_detail_002579.jsp&mid=WC0b01ac058004d5c1 [Google Scholar]
- Gales A.C., Reis A.O., Jones R.N. Contemporary assessment of antimicrobial susceptibility testing methods for Polymyxin B and Colistin: review of available interpretative criteria and quality control guidelines. J. Clin. Microbiol. 2001:183–190. doi: 10.1128/JCM.39.1.183-190.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Giacopello C., Foti M., Mascetti A., Grosso F., Ricciardi D., Fisichella V., Lo Piccolo F. Antimicrobial resistance patterns of Enterobacteriaceae in European wild bird species admitted in a wildlife rescue centre. Vet. Ital. 2016;52:139–144. doi: 10.12834/VetIt.327.1374.2. [DOI] [PubMed] [Google Scholar]
- Gomes A.R., Justino C., Rocha-Santos T., Freitas A.C., Duarte A.C., Pereira R. Review of the ecotoxicological effects of emerging contaminants on soil biota. J. Environ. Sci. Health A. Tox. Hazard Subst. Environ. Eng. 2017;52:992–1007. doi: 10.1080/10934529.2017.1328946. [DOI] [PubMed] [Google Scholar]
- Graham D.W., Collignon P., Davies J., Larsson D.G.J., Snape J. Under appreciated role of regionally poor water quality on globally increasing antibiotic resistance. Environ. Sci. Technol. 2014;48:11746–11747. doi: 10.1021/es504206x. [DOI] [PubMed] [Google Scholar]
- Guenther S., Grobbel M., Lubke-Becker A., Goedecke A., Friedrich N.D., Wieler L.H., Ewers C. Antimicrobial resistance profiles of Escherichia coli from common European wild bird species. Vet. Microbiol. 2010;144:219–225. doi: 10.1016/j.vetmic.2009.12.016. [DOI] [PubMed] [Google Scholar]
- Heuer H., Schmitt H., Smalla K. Antibiotic resistance gene spread due to manure application on agricultural fields. Curr. Opin. Microbiol. 2011;14:236–243. doi: 10.1016/j.mib.2011.04.009. [DOI] [PubMed] [Google Scholar]
- Hume R., Kirwan G.M. Eurasian thick-knee (Burhinus oedicnemus) In: Del Hoyo J., Elliott A., Sargatal J., Christie D.A., De Juana E., editors. Handbook of the Birds of the World Alive. Lynx Edicions; Barcelona: 2013. [Google Scholar]
- Janatova M., Albrechtova K., Petrzelkova K.J., Dolejska M., Papousek I., Masarikova M., Cizek A., Todd A., Shutt K., Kalousova B., Profousova-Psenkova I., Modry D., Literak I. Antimicrobial-resistant Enterobacteriaceae from humans and wildlife in Dzanga-Sangha protected area, Central African Republic. Vet. Microbiol. 2014;171:422–431. doi: 10.1016/j.vetmic.2014.02.014. [DOI] [PubMed] [Google Scholar]
- Järhult J.D., Stedt J., Gustafsson L. Zero prevalence of extended spectrum beta-lactamase-producing bacteria in 300 breeding collared flycatchers in Sweden. Infect. Ecol. Epidemiol. 2013;3 doi: 10.3402/iee.v3i0.20909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keen P.L., Montforts M.H.M.M. Wiley-Blackwell; Hoboken, New Jersey: 2012. Antimicrobial Resistance in the Environment. [Google Scholar]
- Kruse H., Sorum H. Transfer of multiple drug resistance plasmids between bacteria of diverse origins in natural microenvironments. Appl. Environ. Microbiol. 1994;60:4015–4021. doi: 10.1128/aem.60.11.4015-4021.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liofilchem® . 2019. Antibiotic Disc Interpretative Criteria and Quality control—Rev.14.http://www.liofilchem.net/pdf/disc/disc_interpretative_table.pdf [Google Scholar]
- Magiorakos A.P., Srinivasan A., Carey R.B., Carmeli Y., Falagas M.E., Giske C.G., Harbarth S., Hindler J.F., Kahlmeter G., Olsson-Liljequist B., Paterson D.L., Rice L.B., Stelling J., Struelens M.J., Vatopoulos A., Weber J.T., Monnet D.L. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 2012;18:268–281. doi: 10.1111/j.1469-0691.2011.03570.x. [DOI] [PubMed] [Google Scholar]
- Mascara R., Sarà M. Censimento di specie d’uccelli steppico-cerealicole d’interesse comunitario nella Piana di Gela (Sicilia sud-orientale) (Aves) Il Naturalista Siciliano. 2007;31:27–39. [Google Scholar]
- Peronace V., Cecere J.G., Gustin M., Rondinini C. The 2011 Red List of Italian breeding birds. Avocetta. 2012;36:11–58. [Google Scholar]
- Radhouani H., Poeta P., Goncalves A., Pacheco R., Sargo R., Igrejas G. Wild birds as biological indicators of environmental pollution: antimicrobial resistance patterns of Escherichia coli and enterococci isolated from common buzzards (Buteo buteo) J. Med. Microbiol. 2012;61:837–843. doi: 10.1099/jmm.0.038364-0. [DOI] [PubMed] [Google Scholar]
- Radhouani H., Silva N., Poeta P., Torres C., Correia S., Igrejas G. Potential impact of antimicrobial resistance in wildlife, environment, and human health. Front. Microbiol. 2014;5:23. doi: 10.3389/fmicb.2014.00023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ramey A.M., Hernandez J., Tyrlöv V., Uher-Koch B.D., Schmutz J.A., Atterby C., Järhult J.D., Bonnedahl J. Antibiotic-resistant Escherichia coli in migratory birds inhabiting remote Alaska. EcoHealth. 2018;15:72–81. doi: 10.1007/s10393-017-1302-5. [DOI] [PubMed] [Google Scholar]
- Rossney A.S., Shore A.C., Morgan P.M., Fitzgibbon M.M., O'Connell B., Coleman D.C. The emergence and importation of diverse genotypes of methicillin-resistant Staphylococcus aureus (MRSA) harboring the Panton-Valentine leukocidin gene (pvl) reveal that pvl is a poor marker for community-acquired MRSA strains in Ireland. J. Clin. Microbiol. 2007;45:2554–2563. doi: 10.1128/JCM.00245-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rysz M., Alvarez P.J. Amplification and attenuation of tetracycline resistance in soil bacteria: aquifer column experiments. Water Res. 2004;38:3705–3712. doi: 10.1016/j.watres.2004.06.015. [DOI] [PubMed] [Google Scholar]
- Shobrak M.Y., Abo-Amer A.E. Role of wild birds as carriers of multi-drug resistant Escherichia coli and Escherichia vulneris. Braz. J. Microbiol. 2014;45:1199–1209. doi: 10.1590/s1517-83822014000400010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spena M.T., Grasso R., Pollonara E., Giunchi D., Baldaccini N.E. Indagini preliminari sulla biologia riproduttiva e sugli aspetti ecologici dell’Occhione (Burhinus oedicnemus) nella penisola di Thapsos (Siracusa) Biogeographia. 2011;30:651–658. [Google Scholar]
- Spena M.T., Foti M., Fisichella V., Mascetti A., Zafarana M.A., Colnaghi M., Grasso M., Piraino C., Sciurba F., Grasso R. Physiological and potentially pathogenic microbial flora in Stone curlew (Burhinus oedicnemus) in Sicily (Italy) J.Wildl. Biodivers. 2020;4:24–36. [Google Scholar]
- Tinarelli R., Alessandria G., Giovacchini P., Gola L., Ientile R., Meschini A., Nissardi S., Parodi R., Perco F., Taiariol P.L., Zucca C. Consistenza e distribuzione dell’occhione in Italia: aggiornamento al 2008. In: Giunchi D., Pollonara E., Baldaccini N.E., editors. L’occhione (Burhinus oedicnemus): Biologia e conservazione di una specie di interesse comunitario - Indicazioni per la gestione del Territorio e Aree protettte. Consorzio del Parco Fluviale Regionale del Taro, Collecchio. 2008. pp. 45–50. [Google Scholar]
- Tormoehlen K., Johnson-Walker Y.J., Lankau E.W., Myint M.S., Herrmann J.A. Considerations for studying transmission of antimicrobial resistant enteric bacteria between wild birds and the environment on intensive dairy and beef cattle operations. PeerJ. 2019;7 doi: 10.7717/peerj.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vittecoq M., Godreuil S., Prugnolle F., Durand P., Brazier L., Renaud N., Arnal A., Aberkane S., Jean-Pierre H., Gauthier-Clerc M., Thomas F., Renaud F. Antimicrobial resistance in wildlife. J. Appl. Ecol. 2016;53:519–529. [Google Scholar]
- Wellington E.M., Boxall A.B., Cross P., Feil E.J., Gaze W.H., Hawkey P.M., Johnson-Rollings A.S., Jones D.L., Lee N.M., Otten W., Thomas C.M., Williams A.P. The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria. Lancet Infect. Dis. 2013;13:155–165. doi: 10.1016/S1473-3099(12)70317-1. [DOI] [PubMed] [Google Scholar]
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