FEMS Microbiology Ecology, 91, 2015, fiv114 doi: 10.1093/femsec/fiv114 Advance Access Publication Date: 21 September 2015 Research Article
RESEARCH ARTICLE
Changes in soil bacterial communities and diversity in response to long-term silver exposure Sotirios Vasileiadis1,2,∗ , Edoardo Puglisi3 , Marco Trevisan2 , Kirk G. Scheckel4 , Kate A. Langdon5 , Mike J. McLaughlin5 , Enzo Lombi1 and Erica Donner1 1
Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095, Australia, 2 Istituto di Chimica Agraria e Ambientale, Universita´ Cattolica del Sacro Cuore, 29122 Piacenza, Italia, 3 Istituto di Microbiologia, Universita´ Cattolica del Sacro Cuore, 29122 Piacenza, Italia, 4 National Risk Management Research Laboratory, US EPA, Cincinnati, OH 45224, USA and 5 CSIRO Minerals Down Under Flagship, Glen Osmond, SA 5064, Australia ∗ Corresponding author: Centre for Environmental Risk Assessment and Remediation (CERAR), Building X, University Boulevard, University of South Australia, Mawson Lakes, SA 5095, Australia. Tel: +61-88302-2178; E-mail:
[email protected] One sentence summary: High-throughput bacterial DNA sequencing and advanced chemical and spectroscopic techniques revealed complex changes in bacterial abundance and diversity in response to silver-induced selective pressure in soil microbial communities. Editor: Cindy Nakatsu
ABSTRACT Silver-induced selective pressure is becoming increasingly important due to the growing use of silver (Ag) as an antimicrobial agent in biomedical and commercial products. With demonstrated links between environmental resistomes and clinical pathogens, it is important to identify microbial profiles related to silver tolerance/resistance. We investigated the effects of ionic Ag stress on soil bacterial communities and identified resistant/persistent bacterial populations. Silver treatments of 50–400 mg Ag kg−1 soil were established in five soils. Chemical lability measurements using diffusive gradients in thin-film devices confirmed that significant (albeit decreasing) labile Ag concentrations were present throughout the 9-month incubation period. Synchrotron X-ray absorption near edge structure spectroscopy demonstrated that this decreasing lability was due to changes in the Ag speciation to less soluble forms such as Ag0 and Ag2 S. Real-time PCR and Illumina MiSeq screening of 16S rRNA bacterial genes showed β-diversity changes, increasing α-diversity in response to Ag pressure, and immediate and significant reductions in 16S rRNA gene counts with varying degrees of recovery. These effects were more strongly influenced by exposure time than by Ag dose at these rates. Ag-selected dominant OTUs principally resided in known persister taxa (mainly Gram positive), including metal-tolerant bacteria and slow-growing Mycobacteria. Keywords: bacterial diversity; selective pressure; silver; soil
INTRODUCTION Soil microbial communities have been implicated as sources of antimicrobial resistance genes found in pathogenic microorganisms (Silbergeld et al. 2008; Martinez 2009). Yet due to the immense diversity in environmental microbial assemblages
(Schloss and Handelsman 2006), the direct clinical impact of these suspected resistance reservoirs was difficult to conclusively establish prior to the advent of high-throughput sequencing and functional metagenomics. In 2012, Forsberg et al. published compelling evidence signifying recent, direct exchange
Received: 23 December 2014; Accepted: 16 September 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail:
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FEMS Microbiology Ecology, 2015, Vol. 91, No. 10
of multidrug-resistant genes between soil-dwelling bacteria and clinical pathogens of different species, highlighting the pressing need to better understand soil-based resistomes in order to preempt and manage associated risks. Here, we report new insights into the diversity and identity of silver (Ag) tolerant/resistant bacteria in soils. Silver has been used as an antimicrobial agent for centuries but has recently reemerged at the forefront of antimicrobial science amidst growing global concerns about the increasing impacts of resistant pathogens (WHO 2012). For example, recent research has shown that Ag can be used to potentiate and expand the activity of existing antibiotics (Morones-Ramirez et al. 2013), while technological progress in nanotechnology and material science has facilitated the functional incorporation of Ag in an ever-growing list of medical products, ranging from antibacterial wound dressings and bandages to catheters, prostheses and artificial valves and implants (Faunce and Watal 2010; DeVasConCellos et al. 2012; Hooper et al. 2012; De Giglio et al. 2013). Due to its highly marketable antibacterial properties, Ag is also increasingly used as a biocidal agent in mass-marketed everyday consumer products as varied as room fresheners, dietary supplements, washing machines and socks (Benn et al. 2010; Holtz et al. 2012). This rapid and largely unregulated proliferation of antimicrobial (nano-)Ag products indicates that Ag resistance selective pressure and associated effects may be increasing across a diver¨ sity of environments (e.g. Throback et al. 2007; Gottschalk et al. 2009; Sun et al. 2015). Despite this, the recent flurry of environmental Ag research has largely been restricted to acute toxicity studies and little is known about silver-tolerant/resistant phenotypes. Silver resistance has previously been detected in both environmental and clinical isolates (Deshpande and Chopade 1994; Woods, Cochrane and Percival 2009; Sudheer Khan et al. 2011), but research to date has been very limited and largely confined to the resistance evolution mechanism encoded on the conjugally transferable plasmid pMG101, which also confers resistance to mercury, tellurite and several antibiotics (Gupta et al. 2001; Silver 2003; Mijnendonckx et al. 2013). This IncH group plasmid was subject to detailed study following the outbreak of a virulent Ag-resistant Salmonella infection and subsequent closure of a hospital burns unit (Gupta et al. 2001). Other mechanisms potentially contributing to soil microbial selection by Ag (and other metals) may involve lower level genetic resistance mechanisms like the copper/silver cus resistance operons or the loss of porins (Randall et al. 2015) or may be linked to specific microbial growth, nutrition and reproduc´ 1995; Panikov 1999; tion (e.g. r- K- or L-) strategies (Kozdroj ¨ ¨ Langer, Bohme and Bohme 2004). However, in situ profiling of Ag-selected bacterial ecotypes is currently lacking. With little known about the prevalence of Ag resistance in environmental bacteria and its propensity for horizontal gene transfer (HGT), greater understanding of microbial susceptibility, resistance and resilience to Ag is needed to safeguard the beneficial use of antimicrobial Ag products and ongoing innovation in Ag-based medicine. In this study, we investigated the effects of Ag selective pressure on soil bacterial diversity, and explored the traits of selected operational taxonomic units (OTUs) on the basis of their phylogenetic affiliations. Five soils with differing physico-chemical properties were dosed with ionic Ag and incubated for up to 9 months. Synchrotron X-ray absorption near edge Structure (XANES) spectroscopy and diffusive gradients in thin-film (DGT) devices were used to assess Ag speciation and bioavailability. Effects on the soil bacterial communities were assessed using
quantitative real-time PCR (qPCR) and Illumina MiSeq analysis of 16S rRNA amplicons.
MATERIALS AND METHODS Soil collection, spiking and incubation Agricultural top soils (0–10 cm depth) were collected from five locations across Australia (Balaklava, Charleston, Inman Valley, Kingaroy and Port Kenny). The management prior to sampling was mixed cropping at the Balaklava, Port Kenny and Kingaroy sites, and grazing and pasture at the Inman Valley and Charleston sites. Soils were air-dried, homogenized and sieved (
