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Extended-spectrum β-lactamase–producing Enterobacteriaceae among geckos (Hemidactylus brookii) in a Ghanaian hospital

2019; Elsevier BV; Volume: 25; Issue: 8 Linguagem: Inglês

10.1016/j.cmi.2019.04.007

1469-0691

Daniel Eibach, Michael Nagel, Stephan Lorenzen, Benedikt Hogan, Cristina Belmar Campos, Martin Aepfelbacher, Nimako Sarpong, Jürgen May,

Pharmaceutical and Antibiotic Environmental Impacts

The relevance of livestock such as poultry as well as wildlife as reservoirs in the spread and emergence of drug-resistant bacteria has been highlighted [[1]Guenther S. Ewers C. Wieler L.H. Extended-spectrum beta-lactamases producing E. coli in wildlife: yet another form of environmental pollution?.Front Microbiol. 2011; 2: 246Crossref PubMed Scopus (264) Google Scholar]. In sub-Saharan Africa, geckos are ubiquitously prevalent in and around human dwellings. In particular, the hospital environment in low-resource settings, often with poor sanitary facilities and wastewater management, represents an ideal interface for the transmission of antibiotic-resistant microbes between humans and synanthropic wildlife. We aimed to screen geckos within a hospital compound in Ghana for the presence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and to compare the genomes with ESBL-producing isolates collected from hospitalized children and local poultry. In August 2015, faecal samples from 30 geckos, captured in patient rooms and outside the paediatric ward of Agogo Presbyterian Hospital, Agogo, Ghana, were directly plated on two selective MacConkey plates containing 1 mg/L ceftazidime and 1 mg/L cefotaxime. ESBL production was confirmed by combined disc testing. All human (n = 18) and chicken (n = 2) derived ESBL-producing Escherichia coli, isolated between January and June 2015 within the frame of another study [[2]Falgenhauer L. Imirzalioglu C. Oppong K. Akenten C.W. Hogan B. Krumkamp R. et al.Detection and characterization of ESBL-producing Escherichia coli from humans and poultry in Ghana.Front Microbiol. 2019; 9: 3358Crossref PubMed Scopus (54) Google Scholar], served for genomic comparison. Human isolates were collected from stool samples provided by children aged <15 years at admission to Agogo Presbyterian Hospital. Chicken isolates originate from faeces collected within the town of Agogo. Whole-genome sequencing was performed for all ESBL-producing isolates. Genomic DNA was extracted, and a library preparation was performed using the Illumina Nextera XT Library Prep Kit (Illumina, Eindhoven, Netherlands). An average of 2.5M paired short reads were generated on a MiSeq sequencer (Illumina). To determine single nucleotide polymorphisms (SNPs), reads were aligned to the E. coli reference strain ATCC 8739. Phylogeny was calculated using the R package SNPRelate [[3]Zheng X. Levine D. Shen J. Gogarten S.M. Laurie C. Weir B.S. A high-performance computing toolset for relatedness and principal component analysis of SNP data.Bioinformatics. 2012; 28: 3326-3328Crossref PubMed Scopus (1178) Google Scholar]. For all isolates with plasmids harbouring β-lactamase resistance genes, an average of 140 000 long reads were generated using Oxford Nanopore technology (Oxford Nanopore, Oxford, UK). Unicycler was applied to assemble chromosomes and plasmids [[4]Wick R.R. Judd L.M. Gorrie C.L. Holt K.E. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads.PLoS Comput Biol. 2017; 13e1005595Crossref PubMed Scopus (2798) Google Scholar]. From assembled sequences, multilocus sequence typing types, resistance genes, incompatibility groups and point mutations were determined in silico (http://www.genomicepidemiology.org/). Plasmid genome comparisons were performed by progressiveMauve [[5]Darling A.E. Mau B. Perna N.T. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement.PLoS One. 2010; 5: e11147Crossref PubMed Scopus (2639) Google Scholar]. Thirty geckos were caught and identified on the basis of morphologic traits as geckos of the species Hemidactylus brookii. Of the 30 faecal samples, seven ESBL-producing Enterobacteriaceae, including five E. coli, one Citrobacter freundii and one Enterobacter cloacae, were isolated from four geckos (13%) (Table 1). All ESBL-producing Enterobacteriaceae harboured the blaCTX-M-15 ESBL gene, which were all chromosomally inserted apart from one sequence type (ST)-46 E. coli and one ST-78 E. cloacae isolate. Other identified resistance genes are shown in Supplementary Table S1.Table 1Location of β-lactamase resistance genes by sequence typeSequence typeSample IDHost IDSpeciesLocation on chromosomeLocation on plasmidUnknownG453Gecko/01Citrobacter freundiiblaCTX-M-15aExtended-spectrum β-lactamase gene., blaCMY-37blaOXA-138G454Gecko/02Escherichia coliblaCTX-M-15aExtended-spectrum β-lactamase gene.blaTEM-1BH765Human/01E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.blaTEM-1BH766Human/02E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.blaTEM-1B44H458Human/03E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene., blaOXA-146G451Gecko/03E. coliblaCTX-M-15,aExtended-spectrum β-lactamase gene. blaTEM-1BH463Human/04E. coliblaCTX-M-15,aExtended-spectrum β-lactamase gene. blaTEM-1BH466Human/05E. coliblaCTX-M-15,aExtended-spectrum β-lactamase gene. blaTEM-1B78G452Gecko/01Enterobacter cloacaeblaACT-5blaCTX-M-15aExtended-spectrum β-lactamase gene., blaTEM-1B, blaOXA-190H460Human/06E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene., blaOXA-1167H461Human/07E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene., blaOXA-1H462Human/08E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene., blaOXA-1H467Human/09E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.295H464Human/10E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.410G455Gecko/02E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.blaOXA-1C769Chicken/01E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene., blaOXA-1617H468Human/11E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene., blaOXA-1H471Human/12E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene., blaTEM-1B, blaOXA-11706H459Human/13E. coliblaCTX-M-15,aExtended-spectrum β-lactamase gene. blaTEM-1B3018H469Human/14E. coliblaCTX-M-15,aExtended-spectrum β-lactamase gene. blaTEM-1B3268H465Human/15E. coliblaCTX-M-15,aExtended-spectrum β-lactamase gene. blaTEM-1BH470Human/16E. coliblaCTX-M-15,aExtended-spectrum β-lactamase gene. blaTEM-1B6359G456Gecko/04E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.blaTEM-1BG457Gecko/04E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.H767Human/17E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.blaTEM-1BH768Human/18E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.blaTEM-1BC770Chicken/02E. coliblaCTX-M-15aExtended-spectrum β-lactamase gene.blaTEM-1Ba Extended-spectrum β-lactamase gene. Open table in a new tab When comparing the seven ESBL-producing isolates from geckos with 18 human and two chicken strains, four sequence type clusters, consisting of three to five isolates each, were regarded as possible clonal spread events and were therefore investigated for SNPs (Supplementary Fig. S1). Among the ST-6359 cluster (G456/G457/H767/H768/C770), the two gecko E. coli isolates isolated from the same animal (G456/G457) revealed a difference of 79 SNPs, while the gecko isolate G457 differed by 74 and 62 SNPs from the human isolates (H767/H768) and by 87 SNPs to chicken isolate C770. Similarly, within the ST-46 cluster (G451/H463/H466), human isolates H463 and H466 differed by 78 SNPs and 73 SNPs, respectively, from gecko isolate G451. The ST-38 cluster (G454/H765/H766) and ST-410 cluster (G455/C769) appeared less genetically related, with 251 to 1617 SNPs differences between the human and gecko isolates (Table 1). Two gecko isolates harboured blaCTX-M-15 on a plasmid (Supplementary Table S2). In the E. cloacae isolate (G452), blaCTX-M-15 was s located on a plasmid carrying two plasmid replicon types, IncFII/IncFIB, with a size of 166 365 bp. The E. coli isolate (G451) harbours blaCTX-M-15 on a 63 371 bp plasmid of the IncFIB replicon type. Similar IncFIB type plasmids with the same resistance genes were found in a human ST-46 E. coli isolate (H463; 63 552 bp), a human ST-1706 E. coli isolate (H459; 73 343 bp) and without the dfrA4 gene in another human ST-46 E. coli isolate (H466; 60 859 bp) (Supplementary Fig. S2). These data illustrate relatively close genetic relations and similar resistance plasmids among human-, poultry- and gecko-derived strains, suggesting circulation of similar ESBL-producing bacteria and plasmids in these populations in rural Ghana. As a species inhabiting a small territory, it is highly probable that geckos came in contact with antibiotic resistance genes in or near the paediatric ward of the hospital. This could be explained either by environmental contamination of the hospital compound with antibiotics or its degradation products, which select for resistance; or by direct transmission of resistant bacteria from humans, other animals or environmental sources, including meat products. Poor hygiene and poor sanitation may trigger these events. The present data do not allow conclusions to be drawn regarding the direct transmission of pathogens from patients to geckos, or vice versa. Nevertheless, because gecko faeces are omnipresent and are most likely a permanent environmental source of antibiotic-resistant bacteria, geckos may be relevant for hospital hygiene in tropical countries. The funders had no role in study design, data collection and interpretation or the decision to submit the work for publication. All authors report no conflicts of interest relevant to this article. The authors thank all dedicated fieldworkers and study nurses. Further, we thank D. Winter, V. Levermann (Bernhard Nocht Institute for Tropical Medicine, Hamburg), C. Wiafe Akenten and K. Oppong (Kumasi Centre for Collaborative Research in Tropical Medicine, Kumasi, Ghana) for excellent technical assistance; A. Jaeger (Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany) for organizing the data management; and L. Reigl (Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany) for project management. We thank R. Krumkamp, N. Struck, T. Thye (Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany) and S. Herrera-Leon (National Center of Microbiology, Institute of Health Carlos III, Madrid, Spain) for helpful discussion. The work was supported by a grant to the German Center of Infection Research (DZIF), through the German Federal Ministry of Education and Research (BMBF; grant number 8000 201-3). The following is/are the supplementary data to this article: Download .docx (.11 MB) Help with docx files Multimedia component 1 Download .docx (.08 MB) Help with docx files Multimedia component 2Figs2View Large Image Figure ViewerDownload Hi-res image Download (PPT)