Relevance of tcrYAZB operon acquisition for Enterococcus survival at high copper concentrations under anaerobic conditions

Sir,

Copper (Cu) is essential for eukaryotic and prokaryotic cellular functions.¹ Toxic concentrations for microbes can occur during cellular immunological or predation strategies (e.g. macrophages, amoebas) and anthropogenic activities (e.g. animal feed additives, agriculture microbicides) in diverse environments (e.g. manure, soil).^1,2 Cu tolerance (CuT) among bacteria is associated with its efflux from the cell, detoxification or sequestration.¹ Among Enterococcus, the acquired tcrYAZB operon is the most frequently described CuT mechanism.^3–5 It encodes TcrB, an efflux pump of the P_1B-3-ATPase subgroup, which is activated by Cu²⁺ and, to a lesser extent, Cu⁺.⁶tcrYAZB is often adjacent to a gene cluster including a gene encoding a multicopper oxidase, cueO, presumptively involved in the detoxification of Cu⁺ to Cu²⁺ under aerobic conditions (e.g. GenBank number AHWI01000020.1).¹ The tcrB and cueO genes are frequently co-dispersed among unrelated MDR Enterococcus from diverse sources and species (mainly Enterococcus faecium).³

The impact of non-antibiotic compounds in selection of MDR bacteria has been widely discussed. On this topic, the SCENHIR group published a report (http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_021.pdf) stressing the need to develop standard methodologies important for application in biocide tolerance surveillance programmes (Cu included), for providing informative data in biocidal product development or usage, and for helping to establish regulatory policies. To date, few studies have provided data on CuSO₄ tolerance among Enterococcus,^3–5,7–9 which are sometimes difficult to interpret and/or compare due to: (i) the methodology used, which varies among studies or is not described in adequate detail (e.g. agar dilution/microdilution, pH and media storage period, which could affect free Cu availability, inoculum amount or incubation time);^2–5,7 or (ii) an unclear correlation between MIC_CuSO4, tcrYAZB occurrence and/or Enterococcus species (especially non-E. faecium).^3,5,7–9

The method proposed by Aarestrup and Hasman⁹ is the most used to determine the MIC_CuSO4 among Enterococcus.^3,4,7 Considering that TcrB can be partially activated by Cu⁺ and that TcrZ is a Cu⁺ transporter, we hypothesize that MIC_CuSO4 evaluation using the Aarestrup and Hasman⁹ method under a reduced environment (modifying the atmosphere from the classical aerobiosis to anaerobiosis) could identify more accurately a changing phenotype associated with tcrYAZB acquisition by different Enterococcus species. A first proposal for a CuSO₄ tolerance cut-off to differentiate Enterococcus with the tcrYAZB operon is also given.

We studied 225 Enterococcus (1997–2012, mostly MDR; diverse clonal lineages) and considered as WT isolates those lacking the acquired CuT genes tcrB (part of the tcrYAZB operon) and cueO.³ They included 79 tcrB ± cueO⁺ isolates, 16 cueO⁺ isolates and 130 isolates without these CuT genes (Table 1) of diverse species [E. faecium (n = 139), Enterococcus faecalis (n = 47), Enterococcus hirae (n = 20), Enterococcus gallinarum (n = 10), Enterococcus casseliflavus (n = 5) and Enterococcus durans (n = 4)] and sources [animal production setting (n = 150), humans (n = 39), food (n = 26) and hospital sewage/river (n = 10)] (Table 1).³

MIC_CuSO4 (Sigma-Aldrich-C1297) values were determined using in all assays two sets of freshly prepared Mueller–Hinton II agar (bioMérieux) supplemented with 0.25, 0.5, 1, 2, 4, 8, 12, 16, 20, 24, 28, 32 and 36 mM CuSO₄ (pH = 7.2) with incubation (18–20 h) under anaerobiosis (GENbox jar + GENbox anaerobiosis + anaerobic indicator; bioMérieux) and aerobiosis for comparison. A 0.001 mL suspension of 10⁷ cfu/mL bacteria was applied to each plate. The MIC was considered the first concentration without visible growth (negative + positive controls shown in Table 1). In order to understand whether the presence of tcrB in previous screening assays³ corresponded to the full tcrYAZB operon and how this could have an impact on the CuSO₄ tolerance cut-off proposed, we amplified the region between tcrY and tcrB by long PCR in 39 isolates representative of all MIC_CuSO4 values (four isolates with MIC = 20–28 mM were also sequenced for PCR validation) (Table S1, available as Supplementary data at JAC Online).

Under aerobiosis it was more difficult to clearly differentiate between tcrB⁺ and tcrB⁻ isolates (Table 1). E. faecium showed larger differences between tcrB⁺ and tcrB⁻ isolates (MIC₅₀/MIC₉₀ = 32/36 and 16/20 mM, respectively) and E. faecalis the smallest differences (MIC₅₀/MIC₉₀ >36 mM for both genotypes). Although small in number, tcrB⁺ isolates from other species seem to have higher MICs than tcrB⁻ isolates in most cases. Of note is the growth of several E. faecium, E. faecalis or E. hirae in the same CuSO₄ concentrations (mostly in 20 mM; 32 to >36 mM for E. faecalis), independent of tcrB carriage. However, under anaerobiosis an association between tcrB occurrence and highest CuSO₄ tolerance levels was more evident, with most tcrB⁺ isolates showing MIC ≥16 mM and tcrB⁻/cueO⁺ or without genes an MIC ≤12 mM, independent of species, source, MDR profile or clonal lineage.³ The few MIC ≤12 mM–tcrB⁺ isolates did not amplify tcrYAZB, contrasting with MIC ≥16 mM–tcrB⁺ isolates, suggesting a non-functional gene cluster (Table 1 and Supplementary Data).

In summary, it was demonstrated that under anaerobiosis the acquisition of tcrYAZB is a clear advantage for Enterococcus survival at high Cu concentrations. Moreover, tcrYAZB⁺ Enterococcus could be identified using the Aarestrup and Hasman⁹ agar dilution method under anaerobiosis and a CuSO₄ tolerance cut-off ≥16 mM (based on phenotypic/genotypic assays). This approach was also critical in our previous study to differentiate Salmonella with/without the CuT-silECFBAP cluster, which is impossible under aerobiosis.¹⁰ Better awareness of the role of reducing environments (e.g. gut, piggery-waste lagoons) under multiple stressors (e.g. the more toxic Cu⁺, antibiotics) for enhanced survival and selection/persistence of particular CuT MDR strains is needed.

Funding

This work was supported by national funds [Fundação para a Ciência e a Tecnologia (FCT)] through project UID/MULTI/04378/2013. J. M., E. S. and A. R. F. were supported by fellowships of the FTC (grant no. SFRH/BD/77518/2011, SFRH/BD/63955/2009 and SFRH/BPD/96148/2013, respectively). ESCMID Research Grant 2013 supports the work of A. R. F. The Erasmus+ programme supported the stay of J. R. (Strathclyde University, Glasgow, UK) in Portugal. Work in the lab of T. M. C. is funded by the Ministry of Economy and Competitiveness (PI10-02588).

Transparency declarations

None to declare.

Acknowledgements

We wish to thank Dr Lina Cavaco (Division of Microbiology and Risk Assessment, DTU Food, Technical University of Denmark, National Food Institute, Denmark) for the discussion of agar dilution protocols for CuSO₄. We would also like to thank Dr Jill Williams (Department of Genetics of the University of Melbourne, Melbourne, Victoria, Australia) who kindly provided Escherichia coli ED8739 (pRJ1004).

References