In-Depth Genomic Characterization of a Meropenem-nonsusceptible Pseudomonas otitidis Strain Contaminating Chicken Carcass

Tatiana Regina Vieira, Gustavo Enck Sambrano, Núbia Michelle Vieira da Silva, Priscylla Carvalho Vasconcelos, Esther Ferraza Cavinatto de Oliveira, Celso José Bruno de Oliveira, Samuel Paulo Cibulski, Marisa Cardoso

Abstract


Background: The indiscriminate use of antibiotics in food-animal production has a major impact on public health, particularly in terms of contributing to the emergence and dissemination of antimicrobial resistant bacteria in the food-animal production chain. Although Pseudomonas species are recognized as important spoilage organisms in foodstuff, they are also known as opportunistic pathogens associated with hospital-acquired infections. Furthermore, Pseudomonas can play a role as potential reservoirs of antimicrobial resistance genes, which may be horizontally transferred to other bacteria. Considering that cephalosporins (3rd and higher generations) and carbapenems are critically important beta-lactam antimicrobials in human medicine, this study reports the occurrence and genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain recovered from a chicken carcass in Brazil.

Materials, Methods & Results: During the years 2018-2019, 72 frozen chicken carcasses were purchased on the retail market from different regions in Brazil. Aliquots from individual carcass rinses were screened for Extended Spectrum Beta-lactamase (ESBL)-producing bacteria in MacConkey agar supplemented with 1mg.L-1 cefotaxime. Phenotypically resistant isolates were further tested for resistance to other antimicrobials and confirmed as ESBL-producers by means of disk-diffusion method using Müller-Hinton agar. Only one meropenen-nonsusceptible isolate was detected and submitted to whole genome sequencing (WGS) in Illumina Miseq. The strain was identified as Pseudomonas otitidis by local alignment of the 16S rRNA sequence using BLASTn and confirmed by Average Nucleotide Identity (ANI) analysis using JspeciesWS database. Genes encoding for antimicrobial resistance were detected by means of Resfinder and Comprehensive Antibiotic Resistance Database (CARD) databases. The phenotypic non-susceptibility to meropenen was attributed to the gene blaPOM-1. A total of 192 different genes encoding for quorum sensing system, antiphagocytosis, iron uptake, efflux pump, endotoxin and toxin, adherence, and secretion systems were detected by means of Virulence Factor Database (VFDB). Pseudomonas otitidis-pan genome was built using Roary-rapid large-scale prokaryote pan genome analysis using the present strain (K_25) and other two P. otitidis genomes (PAM-1, DSM 17224) publicly available at the NCBI. The core genome analysis of the two human strains resulted in similar percentages.

Discussion: Carbapenems are critically important drugs for human health and bacterial strains resistant to these antimicrobials pose a public health problem. The blaPOM-1 gene harbored by the Pseudomonas otitidis K_25 strain encodes a metallo-beta-lactamase (MBL) conferring resistance to carbapenems. Pseudomonas otitidis was the first confirmed pathogenic Pseudomonas species expressing MBL constitutively in the absence of inducible beta-lactamase genes. Furthermore, the several virulence genes associated with the capacity of the P. otitidis K_25 to colonize, evade the immune system and cause lesions in the human host confirm this strain as a potential opportunistic pathogen contaminating foodstuff. These reinforce the need to address antimicrobial resistance in a One Health perspective, in which resistant bacteria and resistance determinants circulate among environment, animals and humans.


Full Text:

PDF

References


Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S., Prjibelski A.D., Pyshkin A.V., Sirotkin A.V., Vyahhi N., Tesler G., Alekseyev M.A. & Pevzner P.A. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology. 19(5): 455–477.

Bao E., Jiang T. & Girke T. 2014. AlignGraph: Algorithm for secondary de novo genome assembly guided by closely related references. Bioinformatics, 30(12): 319–328.

Bonardi S. & Pitino R. 2019. Carbapenemase-producing bacteria in food-producing animals, wildlife and environment: A challenge for human health. Italian Journal of Food Safety. 8(2): 7956.

Borgianni L., De Lucca F., Thaller M.C., Chong Y., Rossolini G.M. & Docquier J.D. 2015. Biochemical characterization of the POM-1 metallo-β-lactamase from Pseudomonas otitidis. Antimicrobial Agents and Chemotherapy. 59(3): 1755–1758.

Cabanettes F. & Klopp C. 2018. D-GENIES: Dot plot large genomes in an interactive, efficient and simple way. PeerJ. 6: e4958. [Fonte: ]

Clark L.L., Dajcs J.J., McLean C.H., Bartell J.G. & Stroman D.W. 2006. Pseudomonas otitidis sp. nov., isolated from patients with otic infections. International Journal of Systematic and Evolutionary Microbiology. 56(4): 709–714.

Clinical and Laboratory Standard Institute. 2018. Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standard Institute. 2018.

Clinical and Laboratory Standard Institute. 2020. Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standard Institute. 2020. Disponível em: < http://em100.edaptivedocs.net/GetDoc.aspx?doc=CLSI%20M100%20ED30:2020&scope=user>. [Accessed online in March 2020].

Feng K., Li R., Chen Y., Zhao B. & Yin T. 2015. Sequencing and analysis of the Pseudomonas fluorescens GcM5-1A genome: A pathogen living in the surface coat of Bursaphelenchus xylophilus. PLoS ONE. 10(10): e0141515.

Fernández M., Porcel M., De La Torre J., Molina-Henares M.A., Daddaoua A., Llamas M.A., Roca A., Carriel V., Garzón I., Ramos J.L., Alaminos M. & Duque E. 2015. Analysis of the pathogenic potential of nosocomial Pseudomonas putida strains. Frontiers in microbiology. 6: 871. [Fonte: ].

Gurevich A., Saveliev V., Vyahhi N. & Tesler G. 2013. QUAST: Quality assessment tool for genome assemblies. Bioinformatics. 29(8): 1072–1075.

Lee K., Kim C., Young D., Yum J.H., Chung M. & Chong Y. 2011. POM-1 metallo-β-lactamase-producing Pseudomonas otitidis isolate from a patient with chronic otitis media. Diagnostic Microbiology and Infectious Disease. 72: 295-296.

Liu B., Zheng D., Jin Q., Chen L. & Yang J. 2019. VFDB 2019: A comparative pathogenomic platform with an interactive web interface. Nucleic Acids Research. 47(D1): D687–D692.

McEwen S.A. & Collignon P.J. 2018. Antimicrobial resistance: a one health perspective. Microbiology Spectrum. 6(2): ARBA-0009-2017. DOI: 10.1128/microbiolspec.ARBA-0009-2017.

Nishiyama N., Uechi K., Nakamatsu M., Kinjo T. & Fujita J. 2018. Three cases of POM-1 metallo-β-lactamase producing Pseudomonas otitidis isolated from respiratory specimens. Respirology 23: 307–308.

Pang Z., Raudonis R., Glick B.R., Lin T.J. & Cheng Z. 2019. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnology Advances. 37(1): 177–192.

Quinn P.J., Markey B.K., Leonard F.C., Hartigan P.J., Fanning S. & Fitzpatrick E.S. 2011. Enterobacteriaceae. In: Veterinary Microbiology and Microbial Disease. 2.ed. Iowa: Wiley-blackwell, pp.263-286.

Quintieri L., Fanelli F. & Capuyo L. 2019. Antibiotic resistant Pseudomonas spp. spoilers in fresh dairy products: An underestimated risk and the control strategies. Foods. 8(9): E372. [Fonte: ].

Richter M., Rosselló-Móra R., Oliver Glöckner F. & Peplies J. 2016. JSpeciesWS: A web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics. 32(6): 929–931.

Rocha A.J., Barsottini M.R.O., Rocha R.R., Laurindo M.V., Leandro F., Moraes F.L.L. & Rocha S.L. 2019. Pseudomonas aeruginosa: virulence factors and antibiotic resistance genes. Brazilian Archives of Biology and Technology. 62: e19180503. DOI: 10.1590/1678-4324-2019180503.

Simpson J.T., Wong K., Jackman S.D., Schein J.E., Jones S.J. & Birol I. 2009. ABySS: A parallel assembler for short read sequence data. Genome Research. 19(6): 1117–1123.

Stellato G., Utter D.R., Voorhis A., De Angelis M., Eren A.M. & Ercolini D. 2017. A few Pseudomonas oligotypes dominate in the meat and dairy processing environment. Frontiers in Microbiology. 8: 264. DOI: 10.3389/fmicb.2017.00264. [Fonte: ].

Tan H., Zhang Z., Hu Y., Wu L., Liao F., He J., Luo B., He Y., Zuo Z., Ren Z., Peng G. & Deng J. 2015. Isolation and characterization of Pseudomonas otitidis TH-N1 capable of degrading zearalenone. Food Control. 47: 285–290.

Taybali A.F., Coleman G. & Nguyen K.C. 2015. Virulence attributes and host response assays for determining pathogenic potential of Pseudomonas strains used in biotechnology. PLoS ONE. 10(11): e0143604.

Thaller M.C., Borgianni L., Di Lallo G., Chong Y., Lee K., Dajcs J., Stroman D. & Rossolini G.M. 2011. Metallo-Lactamase Production by Pseudomonas otitidis: a Species-Related Trait. Antimicrobial Agents and Chemotherapy. 55(1): 118–123.

World Health Organization. 2018. Antimicrobial resistance (WHO Fact sheet). Geneva: World Health Organization; February. Disponível em: [accessed online in February 2020].

World Health Organization. 2019. OIE list of antimicrobial agents of veterinary importance. Geneva: World Health Organization. Disponível em: . [accessed online in April 2020].

Wong M.H., Chan E.W. & Chen S. 2015. Isolation of carbapenem-resistant Pseudomonas spp. from food. Journal of Global Antimicrobial Resistance. 3(2): 109–114.

Wu J., Jung B.G., Kim K.S., Lee Y.C. & Sung N.C. 2009. Isolation and characterization of Pseudomonas otitidis WL-13 and its capacity to decolorize triphenylmethane dyes. Journal of Environmental Sciences. 21(7): 960–964.




DOI: https://doi.org/10.22456/1679-9216.103176

Copyright (c) 2020 Tatiana Regina Vieira, Gustavo Enck Sambrano, Núbia Michelle Vieira da Silva, Priscylla Carvalho Vasconcelos, Esther Ferraza Cavinatto de Oliveira, Celso José Bruno de Oliveira, Samuel Paulo Cibulski, Marisa Cardoso

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.