Escherichia coli carrying bla_NDM-1 obtained from a migratory penguin (Spheniscus magellanicus) in the Brazilian seacoast

Abstract

The reservoirs for NDM-producing Enterobacterales are increasing, not only in hospitals, but also in the environment and in the community, challenging the therapeutic efficacy of carbapenems. We aimed to characterize an isolate of Escherichia coli harboring the bla_NDM-1 gene recovered from the bloodstream of a penguin (Spheniscus magellanicus) in Southern Brazil. A total of 74 bacterial isolates recovered from arterial blood samples from dead birds were submitted to species identification and antibiotic susceptibility evaluation. One isolate presented resistance to carbapenems (E. coli 89PenNDM) and proved to harbor the bla_NDM-1 gene by multiplex high-resolution melting real-time PCR (PCR-HRM). Conjugation experiments indicated that the bla_NDM-1 was transmissible to E. coli J53. Whole genome sequencing (WGS) confirmed the presence of the bla_NDM-1 gene in a conjugative plasmid (IncA/C₂ plasmid) in both the E. coli 89PenNDM and its transconjugants. The isolate was classified as ST 156 and many other resistance genes (e.g., sul1, sul,2, strA, floR, tet(A)) were identified, all carried in the same IncA/C2 plasmid. This is the first report of bla_NDM-1-producing E. coli isolated from a penguin in the Brazilian seacoast. The presence of a carbapenemase gene in wildlife animals is of concern as they may become reservoirs of multidrug-resistant bacteria and disseminate them to the environment.

Introduction

The emergence of carbapenem-resistant Enterobacterales (CRE) is a major public health problem and its global spread in clinical settings is of great concern worldwide [1, 2]. Carbapenemases are the main determinants of carbapenem resistance, and the New Delhi metallo-β-lactamase-1 (NDM-1) encoded by the bla_NDM-1 gene has drawn particular attention due to its widespread capacity and ability to hydrolyze all beta-lactams except aztreonam [3].

The reservoirs for CRE organisms are increasing, not only in hospitals, but also in the community and in the environment, which critically challenges the therapeutic efficacy of carbapenems [4]. Although only a few studies have reported the presence of CRE from wildlife animals, their presence in food-producing animals and their environment has been demonstrated [5]. However, little is known about their dissemination and potential transmission to humans [6]. Magellanic penguins (Spheniscus magellanicus) inhabit the coastal zones of the extremely south of South America (Argentina, Uruguay, Chile, and Falkland Islands) and migrate to Brazil, in the Atlantic Ocean, or to Peru, in the case of Pacific Ocean, after the reproductive period to search for food [7]. Several reports describing blaNDM-1 in swallows, black kites, storks, or gulls demonstrate that birds may play a role in antibiotic resistance dissemination [5]. The aim of this study was to characterize an isolate of Escherichia coli harboring the bla_NDM-1 gene recovered from the bloodstream of penguin (during a routine beach monitoring project in Southern Brazil).

Materials and methods

The Santos Basin Beach Monitoring Program (Projeto de Monitoramento de Praias da Bacia de Santos, PMP-BS) is one of the monitoring programs required by the Brazilian Federal Environmental Agency (IBAMA) for the environmental licensing process of oil production and transport by the Brazilian Federal Petrol Company (Petrobras) at the pre-salt province. This program collects ill and dead marine birds, turtles, and mammals found in the Brazilian shoreline. Debilitated animals undergo rehabilitation treatment and the dead ones are submitted to complete necropsy. Arterial blood from ill and dead animals are collected and inoculated into blood cultures bottles (Newprov®, Brazil) and the positive bottles are subcultured onto blood agar plates for bacterial identification and further studies. Between 2015 and 2018, a total of 74 bacteria were obtained from blood samples from 45 penguins (S. magellanicus) and 29 seagulls (Larus dominicanus) under aseptic conditions. Among this collection, one isolate presented resistance to imipenem, ertapenem, and meropenem. This bacterial isolate was obtained from a blood culture which blood was collected directly from the heart of the dead bird during the necropsy examination. The isolate was obtained from a S. magellanicus which was found debilitated in a beach of São Francisco do Sul (September 9, 2019) and taken to rehabilitation in Florianópolis, both municipalities from the Brazilian state of Santa Catarina. This penguin was a juvenile male which was trapped in fishing nets and presented dislocation of his left limb. On October 10, 2018, the left pelvic limb and tarsus were amputated. The penguin died on October 25, 2018, and the necropsy was performed on the next day (the carcass was kept under refrigeration between death and necropsy).

For this study, bacterial isolates recovered from arterial blood sampled were submitted to species identification using the API system (bioMérieux, France) and antibiotic susceptibility evaluation by the disk diffusion method as described by the Clinical and Laboratory Standards Institute guidelines (CLSI) [8]. The following antibiotics were tested: imipenem, ertapenem, meropenem, amikacin, ampicillin, amoxicillin + clavulanate, ciprofloxacin, gentamicin, norfloxacin, and tetracycline. Isolates with reduced susceptibility to carbapenems were further evaluated by the Blue-Carba test, a colorimetric assay for carbapenemase detection [9]. Isolates with positive result in the Blue-Carba test were submitted to species identification using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (bioMérieux, France) and to carbapenemase genes (bla_NDM-1, bla_KPC-2, bla_VIM-type, bla_GES-type, bla_OXA-48-like, and bla_IMP-type) detection by multiplex high-resolution melting real-time PCR (RT-PCR) according to Monteiro et al. [10]. Conjugation experiments were performed using Escherichia coli J53 as a recipient and minimal inhibitory concentration (MIC) of antibiotics representative of beta-lactams, aminoglycosides, tigecycline, and chloramphenicol was evaluated by broth microdilution according to the ISO 20776–1, and the results were interpreted according to the CLSI guidelines [8].

The genomes of the E. coli 89PenNDM and its transconjugants were extracted with Wizard SV Genomic DNA purification system (Promega, EUA), the library was prepared with Nextera XT DNA Library Prep Kit (Illumina, EUA), and the sequencing was performed in a MiSeq™ platform (Illumina Inc.) with MiSeq Reagent V2 (500 cycles). The obtained sequences were assembled with SPAdes V.3.9 and annotation was performed in NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [11, 12]. Data were analyzed using the web tools Multi Locus Sequence Typing (MLST) version 2.0, ResFinder version 3.2 and PlasmidFinder version 2.0 from the Centre for Genomic Epidemiology web site (http://www.genomicepidemiology.org) in order to characterize sequence typing (ST), antibiotic resistance mechanisms, and the plasmid Inc types, respectively. The sequence alignment and the study of the bla_NDM-1 genetic environment in IncA/C2 plasmid were performed with Geneious version 9.0.

Results and discussion

Among this 74 bacterial collection, one E. coli isolate obtained from a S. magellanicus presented resistance to imipenem, ertapenem, meropenem, ampicillin, amoxicillin + clavulanate, ciprofloxacin, norfloxacin, and tetracycline. This E. coli presented positive result in the Blue-Carba test and the bla_NDM-1 gene was identified by PCR-HRM. Broth microdilution confirmed that E. coli 89PenNDM was fully resistant to carbapenems and that the transconjugant presented significant increase in the MICs of carbapenems, ceftazidime, and chloramphenicol in comparison to E. coli J53 (Table 1).

Table 1.

Minimal inhibitory concentration (mg/L) of several antibiotics for E. coli 89PenNDM, Transconjugant T89PenNDM, and E. coli J53

Antibiotics	MIC (mg/L)
	E. coli 89PenNDM	Transconjugant T89PenNDM	E. coli J53
Ertapenem Imipenem Meropenem Ceftazidime Gentamicin Tigecycline Amikacin Chloranphenicol	32 8 32  > 256 2 2 8  > 512	16 8 8  > 256 2 0.5 4 256	≤ 0.03 0.5 0.06 0.5 2 0.5 4 8

WGS of E. coli 89PenNDM generated the assembly of 120 contigs, which resulted in an estimated draft genome of 5,174,060-bp length, with a G + C content of 50.7% and a total of 4843 coding sequences. In silico analyses of WGS data indicated that the E. coli 89PenNDM belonged to the sequence type ST156 and confirmed the presence of the bla_NDM-1 gene. The WGS of the transconjugant confirmed that the plasmid harboring the bla_NDM-1 gene belonged to the IncA/C2 incompatibility family. Although it was not possible to cover the length of the plasmid sequence entirely, the sum of the contigs comprising the plasmid was 161,769 bp and, in addition to the bla_NDM-1, the plasmid sequences also carried genes related to resistance to sulfonamides (sul1 and sul2), tetracycline (tet(A)), aminoglycoside (aph(3′)-lb, aph(6)-Id, aph(3′)-VI, aac(6′)-Ib-cr), rifampicin (ARR-3), macrolides (mph(A) and erm(B)), phenicols (floR and catB3), fluoroquinolone (qnrB2), ampicillin (bla_TEM-1), and oxacillin (bla_OXA-1). The E. coli 89PenNDM also carried two other plasmids of the incompatibility groups IncFIC(II) and IncFIB, which were not present in the transconjugant.

This is the first report of the carbapenemase bla_NDM-1 in an E. coli obtained from arterial blood of a penguin (Spheniscus magellanicus) in the Brazilian seacoast. In silico analysis of the WGS data of the E. coli 89PenNDM indicated that the isolate belonged to the sequence type ST156, which was previously described in an NDM-5-producing E. coli strain (ECCRA-119) obtained from a poultry farm in Zhejiang, China, in 2017 [13]. WGS of the E. coli 89PenNDM and its transconjugant indicated that the bla_NDM-1 gene was located in an IncA/C2 plasmid, which is a plasmid that usually carries multiple genes related to antibiotic resistance and are broad host-range vehicles, being commonly identified among animal and clinical bacterial isolates of Enterobacterales worldwide [14–18]. The sequences from the plasmid described in this study share two large regions with the plasmid pRH-1238, a conjugative IncA/C2 plasmid carrying a bla_NDM-1 from a Salmonella enterica serovar Corvallis belonging to ST1541 and isolated from black kites (Milvus migrans) in Germany [14]. The full description of the sequence of the IncA/C plasmid containing the blaNDM-1 gene is of great value due to its dissemination potential. This is additionally emphasized by the broad host range of IncA/C plasmids, allowing replication not only in Enterobacterales but also in other bacterial species, such as Pseudomonas and Photobacterium damselae [17]. The first region (~ 38 Kb with 99% identity) contains plasmid partitioning genes and a restriction-modification system; the second region (~ 38 Kb, with 97% identity) is an antibiotic resistance island carrying several resistance genes: florR, sul2, tetA, aph(3′)-lb, aph(6)-Id, and mph(A). In fact, the bla_NDM-1 was located together with other resistance genes bla_OXA-1, catB3, ARR-3, sul1, aph(3′)-VI, and aac(6′)-Ib-cr. The plasmid also carried the resistance genes qnrB2 and bla_TEM-1, located in unique contigs. Detailed analyses indicated that the bla_NDM-1 gene was flanked downstream by a bleomycin resistance gene, and upstream by an IS-30-like element from the ISAba125 transposase family.

There are no reports characterizing bla_NDM-1 carrying plasmids obtained from birds in Brazil. For this reason, we cannot infer whether this penguin was contaminated in Brazil or somewhere else and brought this plasmid to the country. We believe that the penguin entered in contact with the bacterium while it was trapped in fishing nets as it presented signals of drowning. Therefore, one could consider that the bacterium was acquired in the sea natural ecosystem and not in the captivity during the rehabilitation period. These finds raise concern as they indicate that the bacterium (E. coli) carrying this metalloenzyme is present in the environment and may cause infection (or colonization) of wildlife. Therefore, marine birds may become reservoirs of multidrug-resistant bacteria and, as these animals can travel long distances, may disseminate MDR bacteria in extensive areas of the environment. Continuous surveillance of antimicrobial resistance may significantly contribute to determine possible transmission routes of drug-resistant pathogens between humans, wildlife, and their shared environments.

Author contribution

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Priscila L. Wink, Daiana de Lima-Morales, and Rafael Meurer. The first draft of the manuscript was written by Priscila L. Wink and Daiana de Lima-Morales and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by INPRA — Instituto Nacional de Pesquisa em Resistência Antimicrobiana — Brazil (FIPE/HCPA: 2019–0203, INCT/CNPq: 465718/2014–0 and INCT/FAPERGS: 17/2551–0000514-7).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Declarations

Ethics approval

Animals were collected under ABIO 640/2015, emitted as part of the environmental licensing process required by IBAMA for oil production and transportation by Petrobras in the pre-salt province.

Consent to participate

Not applicable.

Consent to publication

Not applicable.

Conflict of interest

The authors declare no competing interests.