Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal
Abstract
:1. Introduction
2. Materials and Methods
2.1. Studied Areas
2.2. Studied Population and Sample Collection
2.3. Isolation Methodology
2.4. Typing and Antimicrobial Susceptibility Testing (AST)
2.5. Whole Genome Sequencing and Genome Characterization
3. Results
3.1. Detection and Characterization of Isolates
3.2. Serotyping, Virulence Genes, and MLST Analysis
3.3. Antimicrobial Resistance
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Birds Classification | #* | Escherichia coli Isolates | Salmonella Isolates | Campylobacter Isolates | ||||||
Order | Species (Total Samples Analysed) | PCR-Based | WGS-Based | Non-Pathogenic | ||||||
STEC | EAEC | EPEC | ExPEC | |||||||
EVOA | Anseriformes | Anas crecca (17) | 17/17/10 | 1 | 0 | 0 | 0 | 15 | 0 | 0 |
Anas platyrhynchos (1) | 1/1/1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
Ciconiiformes | Ixobrychus minutus (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Gruiformes | Fulica atra (2) ** | 2/2/2 | 0 | 0 | 0 | 0 | 2 | 0 | 2 C. coli | |
Gallinula chloropus (7) ** | 7/7/5 | 0 | 1 | 0 | 1 | 5 | 0 | 1 C. coli/1 C. jejuni | ||
Passeriformes | Acrocephalus schoenobaenus (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Acrocephalus scirpaceus (6) ** | 6/6/6 | 0 | 0 | 0 | 2 | 1 | 0 | 1 C. coli | ||
Cettia cetti (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Carduelis chloris (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Emberiza calandra (3) | 3/3/3 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | ||
Erithacus rubecula (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Euplectes afer (7) | 7/7/7 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Luscinia svecica (6) | 6/6/6 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
Oenanthe oenanthe (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Passer domesticus (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Phylloscopus collybita (4) | 4/4/4 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
Phylloscopus trochilus (7) | 7/7/7 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
Saxicola rubicola (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Strunus unicolor (3) | 3/3/3 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
Total (%) | 71/71/62 | 1 (1.4) | 1 (1.4) | 0 (0) | 3 (4.2) | 30 (42.2) | 0 (0) | 5 (8.1) | ||
CRFPBG | Accipitriformes | Circaetus gallicus (1) ** | 1/1/1 | 0 | 0 | 0 | 0 | 1 | 1 a | 0 |
Ciconiiformes | Ciconia nigra (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Charadriiformes | Larus spp. (18) ** | 18/18/14 | 0 | 0 | 1 | 0 | 10 | 0 | 4 C. jejuni | |
Larus fuscus (1) | 1/1/1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | ||
Fratercula arctica (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Columbiformes | Columba livia (7) ** | 7/7/6 | 1 | 0 | 0 | 0 | 5 | 2 b | 0 | |
Streptopelia decaocto (3) | 3/3/2 | 0 | 0 | 0 | 0 | 3 | 0 | 0 | ||
Falconiformes | Falco peregrinus (1) | 1/1/1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Passeriformes | Garrulus glandarius (1) | 1/1/1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | |
Pelecaniformes | Nycticorax nycticorax (1) | 1/1/0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | |
Strigiformes | Athene noctua (2) | 2/1/1 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | |
Total (%) | 37/37/29 | 1 (2.7) | 0 (0) | 1 (2.7) | 0 (0) | 24 (64.9) | 3 (8.1) | 4 (13.8) | ||
ICBAS | Charadriiformes | Larus spp. (50) *** | 50/0/0 | 0 | 1 | 0 | 6 | 0 | ND | ND |
Total (%) | 50/0/0 | 0 | 1(2.0) | 0 (0) | 6 (12.0) | 0 (0) | ND | ND | ||
Total (%) | 158/108/91 | 2 (1.3) | 2 (1.3) | 1 (0.6) | 9 (5.7) | 54 (34.1) | 3 (2.8) | 9 (9.9) |
EAEC | STEC | EPEC | ExPEC | ||||||||||
O antigen | O92 | O111 | O105 | O45 | O125 ac | O101 | O55 | O78 | O25 | OND | OND | Total (%) | |
H antigen | H33 | H21 | H7 | H2 | H6 | H9 | H10 | H4 | H4 | H16 | H9 | ||
Sequence Type | ST34 | ST40 | ST13581 *** | ST20 | ST583 | ST10 | ST162 | ST23 | ST131 | ST453 | ST410 | ||
# isolates | 1 | 1 ** | 1 | 1 * | 1 | 3 ** | 1 ** | 1 ** | 2 ** | 1 ** | 1 ** | 14 | |
Birds | Acrocephalus scirpaceus | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 2 |
Gallinula chloropus | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 2 | |
Anas crecca | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |
Columba livia | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |
Larus spp. | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 1 | 2 | 1 | 1 | 8 | |
Toxins | astA | 0 | 1 | 0 | 1 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 5 (35.7) |
cea | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 3 (21.4) | |
cvaC | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 3 (21.4) | |
mchF | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 4 (28.6) | |
stx2 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 (14.2) | |
Adhesins | eae | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 3 (21.4) |
Iha | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 2 | 0 | 0 | 5 (35.7) | |
lpfA | 0 | 1 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 1 | 6 (42.9) | |
papC | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 0 | 3 (21.4) | |
yfcV | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 3 (21.4) | |
siderophores | fyua | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 2 | 1 | 1 | 8 (57.1) |
iroN | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 4 (28.6) | |
irp2 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 2 | 1 | 1 | 8 (57.1) | |
iucC | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 2 | 1 | 1 | 8 (57.1) | |
iutA | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 2 | 1 | 1 | 8 (57.1) | |
sitA | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 2 | 1 | 1 | 6 (42.9) | |
Other virulence genes | aggR | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 (14.2) |
chuA | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 3 (21.4) | |
espA | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 3 (21.4) | |
etsC | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 3 (21.4) | |
gad | 1 | 1 | 1 | 1 | 1 | 3 | 1 | 1 | 2 | 1 | 1 | 14 (100) | |
hra | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 3 (21.4) | |
hlyF | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 3 (21.4) | |
iss | 0 | 1 | 1 | 1 | 0 | 3 | 1 | 1 | 2 | 1 | 0 | 11 (78.6) | |
kpsE | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 1 | 0 | 3 (21.4) | |
nleB | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 3 (21.4) | |
OmpT | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 2 | 1 | 0 | 9 (64.3) | |
sat | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 4 (28.6) | |
terC | 1 | 1 | 1 | 1 | 1 | 3 | 1 | 1 | 2 | 1 | 1 | 14 (100) | |
TraT | 0 | 1 | 1 | 1 | 0 | 3 | 1 | 0 | 2 | 1 | 0 | 10 (71.4) |
Bird Species | Pathotype | Serotype/ST | Resistance Phenotype | AMR Determinants (WGS) |
Anas crecca | non-pathogenic | ND | AMP, TET | ND |
Larus fuscus | AMC, AMP | |||
Larus spp. | AMP, TET | |||
Larus spp. | ExPEC | O55:H10/ ST162 | AMP, CAZ, CIP, COX, FEP, NAL, SMX, TET, TMP | blaTEM-1B,blaSHV-12,gyrA, qnrB19,sul2, tet(B), dfrA17 |
ONH:H16/ ST453 | AMP, CAZ, CIP, COX, FEP, NAL, SMX, TET, TMP | blaTEM-1B,blaSHV-12,gyrA, qnrB19,sul2, tet(B), dfrA14 | ||
OND:H9/ ST410 | AMC, AMP, AZM, CAZ, CHL, CIP, COX, FEP, NAL, SMX, TET, TMP | aac(6’)-Ib-cr, mph(A), blaCTX-M-15,blaTEM-1A,blaOXA-1, floR, gyrA, sul1, sul2, tet(A), dfrA17 | ||
O78:H4/ST23 | AMP, CAZ, CIP, COX, FEP, NAL, SMX, TET | blaCTX-M-1, gyrA, sul2, tet(A) | ||
O25:H4/ ST131 | AMC, AMP, AZM, CAZ, CIP, COX, FEP, GMN, NAL, SMX, TET, TMP | aac(6’)-Ib-cr, mph(A), blaCTX-M-15, blaSHV-55, blaOXA-1, aac(3)-IIa, gyrA, qnrS1, sul1, tet(A), dfrA14, dfrA17 | ||
O25:H4/ ST131 | AMC, AMP, AZM, CAZ, CIP, COX, FEP, GMN, NAL, SMX, TET, TMP | aac(6’)-Ib-cr, mph(A), blaCTX-M-15, blaSHV-55, blaOXA-1, aac(3)-IIa, gyrA, qnrS1, sul1, tet(A), dfrA17 | ||
Larus spp. | EAEC | O111:H21/ ST40 | AMC, AMP, CAZ, COX, FOX | ampC-promoter, blaTEM-1B |
Acrocephalus scirpaceus | ExPEC | O101:H9/ ST10 | AMP, CHL, CIP, TET | blaTEM-1B, cmlA1, qnrB19, qnrB82, qnrB67, qnrB56, tet(A) |
O101:H9/ ST10 | AMP, CHL, CIP, TET | blaTEM-1B, cmlA1, qnrB19, qnrB82, qnrB67, qnrB56, tet(A) | ||
Gallinula chloropus | ExPEC | O101:H9/ ST10 | AMP, CHL, CIP, TET | blaTEM-1B, cmlA1, qnrB19, qnrB82, qnrB67, qnrB56, tet(A) |
Columba livia | STEC | O45:H2/ST20 | SMX, TET, TMP | sul1, tet(A), dfrA1 |
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Batista, R.; Saraiva, M.; Lopes, T.; Silveira, L.; Coelho, A.; Furtado, R.; Castro, R.; Correia, C.B.; Rodrigues, D.; Henriques, P.; et al. Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal. Int. J. Environ. Res. Public Health 2023, 20, 223. https://doi.org/10.3390/ijerph20010223
Batista R, Saraiva M, Lopes T, Silveira L, Coelho A, Furtado R, Castro R, Correia CB, Rodrigues D, Henriques P, et al. Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal. International Journal of Environmental Research and Public Health. 2023; 20(1):223. https://doi.org/10.3390/ijerph20010223
Chicago/Turabian StyleBatista, Rita, Margarida Saraiva, Teresa Lopes, Leonor Silveira, Anabela Coelho, Rosália Furtado, Rita Castro, Cristina Belo Correia, David Rodrigues, Pedro Henriques, and et al. 2023. "Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal" International Journal of Environmental Research and Public Health 20, no. 1: 223. https://doi.org/10.3390/ijerph20010223