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This article was downloaded by: [Fekete, Frank A.]On: 7 May 2009Access details: Access Details: [subscription number 910941443]Publisher Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK Geomicrobiology JournalPublication details, including instructions for authors and subscription information: Co-selection of Mercury and Antibiotic Resistance in Sphagnum Core SamplesDating Back 2000 YearsLeslie H. Wardwell a; Brooke A. Jude a; Jennifer P. Moody a; Aaron I. Olcerst a; Ruth A. Gyure b; Robert E.
Nelson c; Frank A. Fekete aa Department of Biology, Colby College, Waterville, ME, United States b Department of Biological andEnvironmental Sciences, Western Connecticut State University, Danbury, CT, United States c Department ofGeology, Colby College, Waterville, ME, United States To cite this Article Wardwell, Leslie H., Jude, Brooke A., Moody, Jennifer P., Olcerst, Aaron I., Gyure, Ruth A., Nelson, Robert E. andFekete, Frank A.(2009)'Co-selection of Mercury and Antibiotic Resistance in Sphagnum Core Samples Dating Back 2000Years',Geomicrobiology Journal,26:4,238 — 247 To link to this Article: DOI: 10.1080/01490450902891854 This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.
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Geomicrobiology Journal, 26:238–247, 2009Copyright Taylor & Francis Group, LLCISSN: 0149-0451 print / 1521-0529 onlineDOI: 10.1080/01490450902891854 Co-selection of Mercury and Antibiotic Resistance
in Sphagnum Core Samples Dating Back 2000 Years
Leslie H. Wardwell,1 Brooke A. Jude,1 Jennifer P. Moody,1 Aaron I. Olcerst,1
Ruth A. Gyure,3 Robert E. Nelson,2 and Frank A. Fekete1
1Department of Biology, Colby College, Waterville, ME 04901, United States
2Department of Geology, Colby College, Waterville, ME 04901, United States 3Department of Biological and Environmental Sciences, Western Connecticut State University, Danbury,CT 06810, United States of about 20 cm (Moore and Webb 1978), at which time mi- Metal exposure might induce multiple drug resistance (MDR)
crobial degradation all but ceases. This produces a finely strat- in bacteria in environments devoid of antibiotics via the process
ified record of vegetational, environmental, geochemical and of co-selection, but the extent is poorly known. Core samples from
chemical change that pollen records indicate displays no more two sphagnum peat bogs in central Maine, USA, were analyzed for
total Hg content and were radiocarbon dated. Culturable bacteria
than 25–40 years of vertical mixing of the record, well within isolated from various core depths were assayed for antibiotic- and
the statistical uncertainties of radiocarbon dating techniques Hg-resistance and the presence of merA (mercuric reductase). Our
(Faegri and Iversen 1975; Moore and Webb 1978; Birks and results show that sphagnum peat bogs represent natural ecosys-
tems that contain ambient levels of Hg that select for indigenous
Species of sphagnum moss are the main constituents of the bacterial strains that are not only Hg resistant, but also possess the
MDR phenotype.
primary mat of ombrotrophic bogs in New England, locally ac-companied by hygrophilous shrubs of the family Ericaceae and Keywords
sedges of the genera Carex and Eriophorum (Cyperaceae). An antibiotic, Co-selection, mercury, resistance, sphagnum ombrotrophic bog is an isolated body of freshwater, with waterand minerals introduced solely through atmospheric deposition.
Until recently, little attention has been given to microbial com-munities of sphagnum bogs. Due to low pH of the substrateand the absence of an adequate nutrient supply, sphagnum moss INTRODUCTION
has been previously considered inhospitable to bacterial growth True peatlands, including sphagnum bogs, are comprised of Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 (Painter 1991). Furthermore, the low buffering capacity in this strictly autochthonous organic matter, partially decayed remains acidic environment enhances the toxicity of metals that may be of plants and other organisms once living at the site, deposited present in sphagnum bogs. However, recent studies have shown layer upon layer in waterlogged situations that allow accumu- that bacterial communities indigenous to sphagnum bogs play lation to exceed decomposition. In sphagnum bogs, permanent crucial nutritional roles in this environment (Opelt and Berg waterlogging occurs once the materials are buried to a depth 2004; Belova et al. 2006; Morales et al. 2006; Opelt et al.
Because sphagnum does not have xylem and has no contact Received 30 October 2008; accepted 11 February 2009.
with soil in a bog setting, free-living nitrogen-fixing organisms C. Kittinger Clark, Bradford Cantor, Samuel Reid, Newton are essential for the nitrogenous requirements and growth of Krumdieck and Elizabeth Littlefield assisted with the core sampling plants (Opelt et al. 2007). High population numbers of dia- in the field. This project was supported at Colby College in part by zotrophic Paenibacillus species have been isolated from sphag- NIH Grant Number P20 RR-016463 from the INBRE Program of the National Center for Research Resources. Additional support at Colby num bogs. In addition, Rahnella spp. have been shown to sol- College came from the Department of Geology (funding for radiocar- ubilize phosphates in the bog environment (Opelt et al. 2007).
bon dating), Department of Biology Honors Research Program (LHW), Although the role that bacterial communities play in sphag- Special Projects Fund, and a Natural Science Division Grant # 01.2303 num mats has been addressed recently, the influence of Hg in Address correspondence to Frank A. Fekete, Department of Bi- the sphagnum bog environment on the co-selection of Hg- and ology, 5729 MH, Colby College, Waterville, ME 04901. E-mail: antibiotic-resistant strains among the indigenous bacterial pop- MERCURY AND ANTIBIOTIC RESISTANCE IN SPHAGNUM BOGS Sphagnum moss has a unique ability to sequester divalent In contrast to these metal polluted environmental test sites, cations (Shotyk et al. 2005; Lodenius et al. 1983). Many heavy sphagnum bogs provide a unique natural biological system for metals, including mercuric ion, exist in the divalent cationic state the investigation of Hg and antibiotic resistance co-selection.
in the environment. Water input to the bog system as either rain Hg in the sphagnum bog environment is present as a natural or runoff does not cause flushing or leaching of these metals from element of the earth’s crust, and in the post Industrial Era, it is the sphagnum substrate; instead they are sequestered tightly by also deposited atmospherically as a pollutant from precipitation the sphagnum, creating an environment rich in toxic and non- (Barkay et al. 2003). Because Hg is held tightly by the sphagnum substrate (Lodenius et al. 1983; Shotyk et al. 2005), it allows Although Hg is pervasive environmentally, many bacterial temporal trends in atmospheric deposition to be observed by species can adapt to its presence by invoking an efficient Hg- examination of Hg levels within the column (Steinnes et al.
detoxification system (Barkay et al. 2003). The most common 2005). Total Hg content analyses of bogs throughout northern bacterial Hg resistance mechanism is reduction of mercuric ion Maine and Ontario (Norton et al. 1997; Givelet et al. 2003; [Hg (II)] to the elemental form [Hg (0)] catalyzed by mercuric Roos-Barraclough et al. 2006) have shown that spikes in Hg reductase (Barkay et al. 2003). The gene encoding mercuric re- concentrations in samples from various core depths correspond ductase, merA, is a part of the mer operon that is often located to times when anthropogenic Hg emissions were high.
on mobile genetic elements such as transposable elements and Cores taken from two bogs, Round Pond bog in Franklin plasmids, and is therefore readily transferred among bacterial County, and Hamilton Pond bog in Kennebec County, Maine, species by horizontal gene transfer (HGT) mechanisms (Sum- USA, have sphagnum mat depths that carbon-date back to ap- mers 2002; Barkay et al. 2003; McIntosh et al. 2008). The mer proximately 2000 ybp (years before present). Total Hg anal- operon is closely linked genetically with antibiotic resistance yses on these bogs revealed ambient Hg concentrations at all genes (Wireman et al. 1997). Antibiotic resistance determinants depths. Bacteria living deep within the core represent popula- are usually organized in gene cassette systems that contain genes tions that originated significantly before the era of antimicrobial conferring resistance to a wide variety of antibiotics (Rowe- chemotherapy. Any selective pressures for antibiotic resistance exerted on microbes indigenous to the acidic environment of a The transfer of antibiotic resistance genes in antibiotic-rich bog that is 2000 years old, were most likely due to co-selection environments such as hospitals, aquaculture and land-based with other non-antibiotic agents such as Hg or other heavy met- agriculture, is a commonly documented phenomenon (Nester als. Other studies have been conducted on metal and antibiotic et al. 1999). However, antibiotic resistance genes are also be- resistance co-selection within metal contaminated sites. Con- ing spread throughout bacterial communities in environments versely, we report here the co-selection of Hg and antibiotic devoid of antibiotics (Baker-Austin et al. 2006). One proposed resistance in a natural environment. Furthermore, this environ- mechanism for the occurrence of antibiotic resistance in such ment contains sufficient Hg to exert selective pressure on indige- environments is via co-resistance, which occurs when the genes nous sphagnum bog bacteria to produce the Hg- and antibiotic- encoding resistance phenotypes are linked together on the same mobile genetic element (Baker-Austin et al. 2006). This phys-ical linkage allows for the co-selection of other genes located Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 on the same genetic element. Associations between heavy metalexposure and specific patterns of antibiotic resistance have been Sampling Site Description and Core Collection
reported (McArthur and Tuckfield 2000; Stepanauskas et al.
Both Hamilton Pond (44◦27 58 N, 69◦50 15 W) and Round Pond (44◦31 14 N, 70◦05 22 W) are kettle basins in complex Linkage between Hg resistance and antibiotic resistance has glacial esker systems, and originated shortly after deglaciation been documented in a wide range of bacterial habitats, including of the region about 14,000 calendar years ago. Round Pond oral and fecal microbial flora of primates (Wireman et al. 1997), bog has a small first-order outlet stream at the southern end; oral microbial flora of patients with amalgam fillings (Summers outflow of excess water from Hamilton Pond is likely subsur- et al. 1993), fish gastrointestinal tracts (Akinbowale et al. 2007), face, through adjoining esker sediments, into adjacent Stuart mine sediments (Nemergut et al. 2004), and freshwater micro- Pond, from which an outlet stream carries excess flow. Neither cosms (Stepanauskas et al. 2006). These bacterial isolates with Hamilton Pond nor Round Pond have inlet streams.
plasmid-encoded mer operons are often shown to be associated A 2 m core from Round Pond bog and a 1 m core from with antibiotic resistance gene cassettes (Mazel et al. 2000).
Hamilton Pond bog were sampled in the fall of 2006 using a However, all of the above studies on co-selection of metal and modified Livingstone Piston Corer. One cm slices of core sedi- antibiotic resistance involve environments either contaminated ments were removed aseptically using sterile spatulas from each with toxic metals or were experimental studies in which metal core at five cm intervals and stored in sealed, sterile polypropy- exposure is directly manipulated to test for co-selection in bac- lene tubes. Sample data were recorded and samples numbered beginning with the oldest (deepest) core samples as the lowest numbered samples and proceeding sequentially to the top of the representing the complete range of core depths, checked for core. Representative samples were selected for evaluation from purity and maintained on stock culture TSA slants. A total of 29 a range of depths in each core. From Hamilton Pond bog, slice metabolically active, or readily culturable isolates were stored numbers 1, 5, 9, 13, 16, and 20, and from Round Pond bog, slice numbers 1, 8, 17, 25, 31, and 36, were processed and used in Bog cores collected in October 2007 were used for recov- this study. Cores were kept wrapped in foil, and both cores and ery of bacterial isolates that were in the endospore stage of samples were stored at 4◦C. The two cores collected in 2006 development at the time of sampling. A sample of 0.5 g of were used for radiocarbon dating and isolation of metabolically sphagnum sample from appropriate slices was added to 4.5 ml active bacterial isolates. Both bogs were cored and re-sampled sterile water. Samples were mixed by vortexing for 5 min and a second time in fall, 2007. Samples from the most recent cores placed in a waterbath at 70◦C for 30 min in order to kill all were analyzed for total Hg concentration and used for isolation metabolically active and non-sporulating bacterial cells. After of endospore-forming bacterial isolates.
appropriate dilution, samples were inoculated and spread platedonto TSA. Cultures were incubated at 20◦C for 72 hours. In- Radiocarbon Dating
dividual colonies were selected from each plate and inoculated Sphagnum bog environments represent sites of slow but onto stock culture TSA slants. A total of 25 endospore-forming continuous surface accumulation of organic matter with neg- isolates were maintained. All isolates were cryogenically stored ligible vertical mixing, so radiocarbon dating of thin slices of at −80◦C in a 50% mixture of glycerol and tryptic soy broth the organic mat (excluding any identifiable modern roots when present) is a standard geological technique for determining agesof deposits. Though recent papers have suggested 14C dating Hg Minimum Inhibitory Concentrations (MICs)
problems with such ombrotrophic peats (e.g., Kilian et al. 1995; Hg minimum inhibitory concentration (MIC) assays were Shore et al. 1995; Nilsson et al. 2001), Blaauw et al. have been performed on each isolate according to the method of Wang, unable to corroborate those findings (Kilian et al. 1995; Shore et et al. (1989). Isolates were grown for 24–48 h at 20◦C on TSA al. 1995; Nilsson et al. 2001; Blaauw et al. 2004). In our study, solid-plating media, and then resuspended a density equivalent 5-mm-thick slices of pure sphagnum peat from approximately to a 2.0 McFarland standard (Wang et al. 1989). After vortexing mid-level and the base of each core were oven-dried at 50◦C each cell suspension thoroughly, 5 µl of each was spotted onto and submitted for commercial AMS radiocarbon dating (Beta TSA plates supplemented with 0 µM, 50 µM, 100 µM, and 250 µM HgCl2. Plates were observed every 24 h for 72 hours,and the MIC was recorded as the lowest concentration of HgCl2 Total Mercury Analysis of Cores
Total mercury concentration (total-Hg) was measured by thermal decomposition, amalgamation and atomic absorption Antibiotic Susceptibility Testing
spectroscopy using a DMA-80 mercury analyzer (Milestone, The activity of antimicrobial agents against all 54 isolates Inc.). Sphagnum samples from Round Pond bog (depths of 1, was assessed in vitro. Antibiotic MIC values for each iso- 6, 101, and 193 cm below the surface) and Hamilton Pond bog Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 late were determined on Sensititre R dried susceptibility panels (depths of 1, 8.5, 50, and 94 cm below the surface) were ana- GN2F (Gram-negative) and GPN2F (Gram-positive) (Trek Di- lyzed. All sphagnum core samples were taken from the center of agnostic Systems, Westlake, OH, USA). Quality control strains wet cores and dried at 40◦C for at least 24 h (or until dry weight Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC stabilized). Six replicate sample aliquots of approximately 0.1g 27853, and Enterococcus faecalis ATCC 29212 were inoculated (mostly leaf with some stem) were prepared from each core in parallel to the isolates as suggested by the manufacturer. The depth and site, and mean total-Hg concentration in µg/kg dry guidelines set by the National Committee for Clinical Labora- weight is reported in Table 3. Commercial greenhouse sphag- tory Standards were followed, with the exception that the MIC num was used for comparison, and a sample of plant material panels were incubated for 72 h at 22◦C.
grown in Hg-free soil was also included as a control.
Strains of Rahnella aquatilis, which were not culturable using the Sensititre R dried susceptibility panel culture sys- Bacterial Strain Isolation, Culture Conditions,
tem, were assayed using the antimicrobial disk susceptibility and Stock Maintenance
test method (Sensi-Discs; Becton, Dickinson and Co., Sparks, First, 1.0 g of sphagnum sample was added to 9.0 ml of sterile MD, USA) containing sulfisoxazole 2 mg, trimethoprim 5 µg, phosphate buffer diluent (Hardy Diagnostics, Santa Monica, piperacillin 100 µg, amikacin 30 µg, tobramycin 30 µg, van- CA. USA). All samples were shaken for fifteen min, serially comycin 30 µg, kanamycin 30 µg, novobiocin 30 µg, peni- diluted and plated onto tryptic soy solid-plating media (TSA, cillin 10 IU/IE/UI, streptomycin 10 µg, and tetracycline 30 µg.
Difco-BBL, Sparks, MD, USA). Culture plates were incubated The 24-hour TSA plating cultures were resuspended to a 0.5 at 20◦C for 72 h. Individual colonies were selected from samples McFarland standard, and were spread by cotton swab evenly MERCURY AND ANTIBIOTIC RESISTANCE IN SPHAGNUM BOGS over TSA plates. After the inocula absorbed into the media, quencing Kit and an ABI Prism 3130 Genetic Analyzer (Applied Sensi-Discs were deposited aseptically onto the culture plates.
Biosystems, Foster City, CA, USA). GenBank database searches Plates were incubated at 20◦C for 2 d prior to measuring zones were carried out for all sequences using the National Center for of inhibition according to the manufacturer’s instructions.
Biotechnology Information basic local alignment search tool(BLAST) web server (
Antibiosis Assays
All metabolically active bacterial isolates were tested for PCR amplification of merA
their ability to produce compounds capable of inhibiting Degenerate primers (Table 1) (Vetriani et al. 2005; Ni Chad- growth of 4 test bacterial strains, (Pseudomonas aeruginosa hain et al. 2006) in 2 sequential PCR experiments were used ATCC7853, Enterococcus faecium ATCC51559, Bacillus sub- to probe all isolates for the merA gene. Genomic DNA was tilis 168 ATCC23857, and Escherichia coli K12 ATCC29425).
extracted as previously described. The first PCR (primers A5 The 24-hour cultures were streaked in 2 parallel straight lines and A2NF; Table 1) generated an amplicon of approximately across TSA solid-plating media. Plates were incubated at 22◦C 1200 bp that was not visible on an agarose gel. Cycling condi- for 3 d. Each of the 4 test strains were resuspended in sterile tions were as follows: 1 cycle of 95◦C for 5 min, 35 cycles of water to a 2.0 McFarland standard and streaked perpendicularly 95◦C for 30 sec; 55◦C for 30 sec; 72◦C for 1 min, followed by from the edge of the plate in the direction of, but not touching, an extension step of 72◦C for 7 min prior to storage at 4◦C. PCR the isolate growth. Plates were incubated at 22◦C for 2 d and using the previous amplicon as a template, and A5 and A1SF isolates were assessed for antimicrobial activity as visualized primers (Table 1), resulted in a 288 base-pair product, which by inhibition of growth of test strains within 5–10 mm of the was confirmed using agarose gel electrophoresis. Cycling con- ditions for the second merA PCR were as follows: 1cycle of95◦C for 5 min, 40 cycles of 95◦C for 30 sec; 56◦C for 20 sec; Bacterial Isolate Identification (16S rRNA Gene
72◦C for 1 min, followed by an extension step of 72◦C for 7 min Sequencing)
Bacterial strains were grown at 20◦C for 1–2 d on TSA. Total genomic DNA was extracted from all bacterial strains using the Colony Fast-ScreenTM kit (Epicentre Technologies, Madison, Radiocarbon age determinations on submitted core samples WI, USA). A single colony was selected from each plate and sus- show clearly that both the Hamilton Pond bog and Round Pond pended in 50 µl of PCR-lyseTM buffer (Colony Fast-ScreenTM bog cores penetrated to sediment depths predating European kit, Epicentre Technologies, Madison, WI, USA). The samples influence (Table 2). The pollen record from our cores also in- were incubated in an iCycler Thermal Cycler (Bio-Rad Labora- dicates minimal mixing at most, and is consistent with these tories, Hercules, CA, USA) at 99◦C for 10 min and were then radiocarbon age determinations, indicating that the cores ex- cooled to 4◦C prior to PCR amplification. PCR amplification tend to well before European contact (Clark CK, Krumdieck cycles conducted with 27f and 1392r primers (Table 1) (Lane NW, Littlefield EF, Nelson RE. 2007. Pre- and Post-European 1991) were as follows: 1 cycle of 94◦C for 3 min, 30 cycles of Forest Composition and Bog Flora Changes Documented by Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 94◦C for 1 min; 55◦C for 1 min; 72◦C for 2 min, followed by a Pollen from Maine Sphagnum Cores. Geol Soc Am Abstracts final extension step of 72◦C for 7.5 min prior to storage at 4◦C.
16S rRNA gene sequencing was performed to confirm the Round Pond bog sphagnum at 175 cm depth was deposited identity of all strains after cleaning the PCR products (E.N.Z.A.
between 325 BCE (before the common era) and 125 BCE, while Cyclepure Kit; Omega Bio-tek, Doraville, GA, USA). A region, sphagnum at 90 cm depth in Hamilton Pond bog was living on approximately 1.5 kb, of the 16S rRNA gene was amplified in all the bog surface about 260 AD. Average sphagnum accumulation isolates using the 27f and 1392r primers (Table 1). Amplicons rates were thus 1.2 mm/yr at Round Pond bog and 0.56 mm/year were sequenced using the Big Dye R Terminator v3.1 Cycle Se- Radiocarbon dates for core samples from Round Pond and Antibiosis assay of Hamilton Pond and Round Pond isolates producing antimicrobial compounds in vitro 325 BCE or 125 BCE Bacillus weihenstephanensis Hg minimum inhibitory concentration (MIC) assays revealed that all metabolically active isolates were resistant to levels of Total Hg concentration analysis of samples from both bogs at least 50 µM HgCl2 (Table 5). Two isolates of Pseudomonas revealed that detectable levels of total Hg were present at all putida (RP23 and RP25) collected from the deepest depth of depths in both Hamilton Pond and Round Pond bogs (Table 3).
the Round Pond bog core showed much higher resistance to Total Hg levels of Round Pond bog ranged from 8.7 parts per Hg. P. putida RP23 grew on TSA supplemented with 100 µM billion (ppb) (at a depth of 193 cm) to 44.9 ppb (at a depth of 6 HgCl2, and P. putida RP25 was resistant to 250 µM HgCl2.
cm). The Hg concentration range was slightly higher at Hamilton Regarding the endospore-forming isolates that were obtained Pond bog where total Hg levels were between 13.1 ppb (at the after sphagnum sample heat treatment, resistance to Hg was not sphagnum mat surface) and 88 ppb (at a depth of 90 cm).
as high (Table 6). Seventeen of the 25 endospore-forming strains Of the 29 bacterial isolates assayed for antibiotic producing were inhibited by a concentration of 50 µM HgCl2. All other activity, only 3 strains inhibited growth of test bacterial strains isolates were inhibited by 100 µM HgCl2.
(Table 4). Both Pseudomonas putida HB11, isolated from a The antibiogram showing only maximal MIC’s for each an- core depth of 55 cm, and Paenibacillus spp. HB26, isolated timicrobial compound tested on each isolate is illustrated in from a core depth of 20 cm, in Hamilton Pond bog core sam- Tables 5 and 6. Regarding those strains that were immediately ples, inhibited growth of the test organism B. subtilis 168. The culturable from the sphagnum samples, termed the metaboli- Pseudomonas putida HB11 strain also inhibited growth of E. cally active isolates, multiple drug resistances were observed in faecium. Bacillus weihenstephanensis RP7, isolated from a core each sample taken throughout the depth of the core. Bacterial depth of 30 cm in Round Pond bog inhibited growth of E. fae- resistance to antimicrobials in the ß-lactam, aminoglycoside, cium and E. coli K12 (Table 4).
cephem and folate pathway inhibitor families was prevalentin both bog core samples. Overall, the endospore-forming iso- lates were more susceptible to antibiotics than the metabolically Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 Total Hg concentration of dry leaf and stem Sphagnum core active isolates; 26% of the endospore-forming strains were sus- ceptible to all antibiotics tested, and 21% isolates were maxi-mally resistant to only 1 antibiotic (Table 6).
Fifty-three of 54 bacterial isolates were identified using NCBI BLASTn, and the results are illustrated in Tables 5 and 6.
Forty-five of the isolates generated BLASTn homologs allow- ing identification at the species level. Sequencing of 16S rRNA gene amplicons revealed high species diversity in each bog. Rah- nella, Paenibacillus, Pseudomonas and Bacillus were the most highly represented genera of the metabolically active isolates.
Eleven of the 29 metabolically active isolates were identified as Rahnella aquatilis and 7 were identified as Paenibacillus spp. As expected, all endospore-forming isolates were Gram- positive. Eleven of the 25 endospore-formers belong to the genus Paenibacillus; other representative genera capable of sproula- tion include Bacillus, Lysinibacillus, Cohnella and Sporosarcina Depths noted represent the distance below surface in which cores were sampled. For each sample, n = 6. ∗greenhouse Sphagnum; Amplification of merA using published primer sets produced ‡Hordeum vulgare, barley in non-Hg soil.
the expected product of 288 base pairs (data not shown). Of the MERCURY AND ANTIBIOTIC RESISTANCE IN SPHAGNUM BOGS Characterization and antibiogram of metabolically active bacterial strains isolated from Round Pond and Hamilton Pond bogs Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 Antibiotic MIC values for each isolate were determined on Sensititre R dried susceptibility panels GN2F (Gram-negative) [amikacin (AMI), ampicillin (AMP), aztreonam (AZT), cefazolin (FAZ), cefepime (FEP), cefotetan Na (TANS), ceftriaxone (AXO), ceftazidime (TAZ), cefoxitin(FOX), cefuroxime (FUR), ciprofloxacin (CIP), gentamicin (GEN), imipenem (IMI), gatifloxacin (GAT), meropenem (MERO), piperacillin(PIP), nitrofurantoin (NIT), piperacillin/tazobactam constant 4 (P/T4) ticarcillin / clavulanic acid constant 2 (TIM2) tobramycin (TOB),trimethoprim/Sulfamethoxazole (SXT), cefpodoxime (POD), ampicillin/sulbactam 2:1 ratio (A/S2)] and sentititre disks sulfisoxazole (SUL)2 mg, trimethoprim (TMP) 5 µg, piperacillin (PIP) 100 µg, amikacin (AMK) 30 µg, tobramycin (TOB) 30 µg, vancomycin (VAN) 30 µg,kanamycin (KAN) 30 µg, novobiocin (NOV) 30 µg, penicillin (PEN) 10 IU/IE/UI, streptomycin (STR) 10 µg, and tetracycline (TET) 30 µg.
*Core depth in centimeters below surface.
Characterization and antibiogram of endsopore-forming bacterial strains isolated from Round Pond and Hamilton Pond bogs Antibiotic MIC values for each isolate were determined on Sensititre R dried susceptibility panels GPN2F (Gram-positive) [erythromycin (ERY), clarithromycin (CLA), vancomycin (VAN), quinupristin/dalfopristin (SYN), clinadamycin (CLI), cefazolin (FAZ), tetracycline (TET),ampicillin (AMP), gentamicin (GEN), levofloxacin (LEVO), linezolid (LZD), ceftriaxone (AXO), streptomycin (STR), penicillin (PEN), ri-fampin (RIF), gatifloxacin (GAT), ciprofloxacin (CIP), trimethoprim/sulfamethoxazole (SXT), oxacillin + 2% NaCl (OXA+)] (Trek Di- agnostic Systems, Westlake, OH) or sentititre disks sulfisoxazole (SUL) 2 mg, trimethoprim (TMP) 5 µg, piperacillin (PIP) 100 µg,amikacin (AMK) 30 µg, tobramycin (TOB) 30 µg, vancomycin (VAN) 30 µg, kanamycin (KAN) 30 µg, novobiocin (NOV) 30 µg, peni- Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 cillin (PEN) 10 IU/IE/UI, streptomycin (STR) 10 µg, and tetracycline (TET) 30 µg. # NMR: no maximal resistance observed. ∗Core depth ismeasured in cm below surface.
29 metabolically active isolates from Round Pond bog, merA verse indigenous bacterial species (Opelt and Berg 2004; Belova was detected in 73% of the isolates. The percentage of merA- et al. 2006; Morales et al. 2006; Opelt et al. 2007). However, positive isolates found at Hamilton Pond bog was comparable at this study incorporates both of the above research areas in or- 67% (Table 5). There were markedly fewer endospore-forming der to investigate the characteristics of the bacterial response isolates that had detectable merA gene amplicons. The percent- to sequestered Hg in the sphagnum environment. We show that age of merA positive endospore-forming isolates was 28% for sphagnum bogs represent a novel system in which to study the Round Pond bog and 29% for Hamilton Pond bog. Table 6 co-selection of antibiotic and Hg resistant bacterial strains that shows that the distribution of detectable merA amplicons in the sporulating sample set does not correlate with bog depth.
The bacterial species isolated from Round Pond and Hamil- ton Pond bogs show high diversity. Pseudomonas, Rahnellaand Paenibacillus are the most common genera observed in the DISCUSSION
readily culturable or metabolically active strain set (Table 5).
Separate studies have been conducted focusing on sphag- Previous studies on sphagnum bogs have shown high popula- num’s sequestration properties for Hg (Lodenius et al. 2003; tion levels of Rahnella spp., and these species are postulated to Shotyk et al. 2005) and the characteristics of sphagnum bogs’ di- play a role in the solubilization of phosphates (Opelt et al. 2007).
MERCURY AND ANTIBIOTIC RESISTANCE IN SPHAGNUM BOGS As free-living diazotrophs, Paenibaccillus spp. fix atmospheric lates were able to produce compounds such that growth was nitrogen that is critical for the growth of the moss and other inhibitory to the antibiosis test bacterial strains (Table 4). Stud- plants indigenous to the ombrotrophic sphagnum bog system ies previously conducted on bacterial communities of sphagnum bogs have found similar low incidences of antimicrobial produc- Not unexpectedly, Pseudomonas spp. were prevalent at all ing bacteria (Opelt et al. 2007). A low incidence of bacteria able depths of the core samples because Pseudomonas spp. are ubiq- to produce antibiotics in the bog environments suggests that uitous environmentally by virtue of their catabolic diversity.
not only is the deep sphagnum substrate free of anthropogenic Previous studies on microorganisms commonly associated with antibiotic influence, but it also does not contain high levels of sphagnum bogs have shown that these species predominantly natural antibiotics. Accordingly, bacterial isolates from deep attach to plant surfaces, and as a consequence do not migrate subsurface sphagnum do not have a functional need for genetic vertically in sphagnum bog columns (Hasebe et al. 2003). Bacte- resistances to broad classes of antibiotics because of the appar- rial communities living in aquatic or water-logged environments ent absence of selection pressure exerted by antibiotics in this like sphagnum bogs tend to form biofilms, which enable a more efficient and protected static mode of cellular growth in com- Despite the apparent absence of natural and anthropogenic parison to planktonic cells in these environments (Davey and antibiotic selective pressures, MDR bacteria were isolated from all depths of the sphagnum cores in both bogs (Tables 5 and Mercury analysis in the current study revealed detectable 6). Broad maximal resistances to antibiotics in the ß-lactam, levels of total Hg at all depths of both sphagnum cores (Table aminoglycoside, cephem and folate pathway inhibitor families 3). The Hg ranges observed at Round Pond (8.7–44.9 ppb) and were observed. Pseudomonas spp. demonstrated the greatest Hamilton Pond (13.1–88 ppb) bogs are consistent with total Hg antibiotic resistance withP. putida strains RP 4 and RP 5 pos- concentrations found in other sphagnum bogs located within the sessing maximal resistance to 13 of the 23 tested antibiotics. Of same geographic range (Norton et al. 1997; Givelet et al. 2003; the gram-positive isolates, Paenibacillus sp. RP 16 exhibited the Roos-Barraclough et al. 2006). Although Hg is currently con- greatest antibiotic resistance to 11 of the 19 tested antibiotics sidered a common environmental contaminant, it is also a heavy (Table 5). Only 3 gram-positive metabolically active isolates metal that exists as an element in the earth’s crust (Barkay et al.
had resistance to the fluorquinolone, ciprofloxacin (Table 5).
2003). Because Hg is prevalent throughout the bog core sam- However, endospore-forming isolates possessed ciprofloxacin ples, and is held tightly in its divalent cationic form by sphagnum resistance (Table 6). In addition, Paenibacillus spp. RP14 and substrate (Lodenius et al. 2003; Shotyk et al. 2005), sphagnum RP 16 show resistance to the macrolides erythromycin and clar- bogs represent an environment that is relatively high in bio- ithromycin as well as the glycopeptide vancomycin (Table 5).
logically available Hg. Because of mercury’s inherent toxicity, When many diverse species of bacteria isolated from the same bacterial species indigenous to this environment must adapt to environment have similar multiple drug resistance phenotypes, the presence of this metal as a means of survival.
resistance genes are commonly found to be encoded on mobile Round Pond bog and Hamilton Pond bog are both ancient genetic elements such as plasmids and transposable elements sphagnum environments. Radiocarbon dating (Table 2) of se- (Guerra et al. 2001; Rowe-Magnus and Mazel 2002; Tennstedt lected samples from these two bogs shows average annual Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 growth of 1.2 mm/yr at Round Pond bog and 0.56 mm/yr at Also located on plasmids and certain transposable elements Hamilton Pond bog. Radiocarbon dates suggest that sphagnum are genes for Hg resistance, including the structural genes lo- moss deep below the surface of the bog is contained in an ancient cated in the mer operon (Hobman et al. 2002; Barkay et al.
environment, yet we observed that bacterial strains isolated at 2003). Interestingly, the metabolically active bacterial strains these depths exhibited multiple drug resistance. Dates for de- exhibited high levels of Hg resistance, suggesting that ambient position of sphagnum at the base of cores taken from Round concentrations of Hg in the sphagnum bogs were at sufficiently Pond bog and Hamilton Pond bog, 325BCE and 260AD (Table high levels to function as a selective agent in this environment 2) respectively, are well before the application of antimicrobial (Table 5). The Hg-resistance genotype, as detected by the pres- chemotherapy in the 1940s. Therefore, high levels of antibiotic ence of merA amplicons, was observed in the majority of the resistance observed in bacterial populations far below the bog metabolically active bacterial isolates (73% in Round Pond bog surface are not a result of selection that was influenced anthro- and 67% in Hamilton Pond bog). Though it is likely the re- pogenically, since these bacteria have not likely been exposed maining metabolically active isolates also carry merA based on to high levels of manufactured antimicrobial compounds.
phenotypic observation of mercury resistance, the high diversity Many antimicrobial compounds, e.g., antibiotics, although of merA confounds efforts to design PCR primer sets to univer- commonly associated with use in the medical and veterinary sally amplify the gene (Barkay et al. 2003). Bacterial strains industries, are produced as secondary metabolites by microor- possessing the mer operon were distributed evenly at all depths ganisms in the environment. An antibiosis assay was performed of both cores, which suggests that biologically available Hg on all readily culturable or metabolically active strains isolated concentrations throughout the sphagnum cores were sufficient from Round Pond and Hamilton bogs. Only 10% of these iso- to influence selection of Hg-resistant bacterial strains.
The observation of the presence of merA amplicons in the to co-select indigenous bacterial strains possessing Hg- and majority of the isolates from Round Pond and Hamilton Pond multiple drug resistance phenotypes.
bogs, both of which are environments with apparently low levelsof antibiotics, provides support for the co-selection hypothesis.
In these locations sphagnum-sequestered Hg is selecting forbacterial strains possessing the mer operon (McIntosh et al.
2008). Because of the mechanism of co-resistance in which Akinbowale OL, Peng H, Grant P, Barton MD. 2007. Antibiotic and heavy metal resistance in motile aeromonads and pseudomonads from rainbow mer operons and antibiotic resistance gene cassettes are linked trout (Oncorhynchus mykiss) farms in Australia. Int J Antimicrob Agents proximally on transposable elements and plasmids (Mazel et al.
2000; McArthur and Tuckfield 2000; Nemergut et al. 2004), Hg Baker-Austin C, Wright MS, Stepanauskas R, McArthur JV. 2006. Co-selection in these bog environments would also provide selection pressure of antibiotic and metal resistance. Trends Microbiol 14:176–182.
for bacterial strains with multiple drug resistance determinants.
Barkay T, Miller SM, Summers AO. 2003. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol Rev 27:355–384.
The endospore forming bacterial isolates provide additional Belova SE, Pankratov TA, Dedysh SN. 2006. Bacteria of the genus Burkholderia support for the co-selection of Hg and antibiotic resistant strains as a typical component of the microbial community of sphagnum peat bogs.
in the sphagnum bog environment. Certain Gram-positive bacte- rial species have the ability to develop into a dormant state with Birks HJB, Birks HH. 1980. Quaternary palaeoecology. Baltimore: University the formation of endospores in highly stressed environments or Blaauw M, van der Plicht J, van Geel B. 2004. Radiocarbon dating of bulk peat in conditions not conducive to vegetative growth (Driks 2002).
samples from raised bogs: non-existence of a previously reported “reservoir The endospore forming strains isolated from both Hamilton effect”? Quat Sci Rev 23:1537–1542.
Pond and Round Pond bogs show generally low resistances Davey ME, O’Toole GA. 2000. Microbial biofilms: from ecology to molecular to Hg, with the majority of isolates having MIC’s of 50 genetics. Microbiol Mol Biol Rev 64:847–867.
Driks A. 2002. Overview: Development in bacteria: spore formation in Bacillus 2 (Table 6). The frequency of merA amplicons detected subtilis. Cell Mol Life Sci 59:389–391.
in sporulating strains (29% of Hamilton Pond bog isolates and Faegri K, Iversen J. 1975. Textbook of pollen analysis. New York: Hafner Press.
28% of Round Pond bog isolates) in comparison to the readily culturable and metabolically-active isolates was markedly less.
Givelet N, Roos-Barraclough F, Shotyk W. 2003. Predominant anthropogenic Correspondingly, overall antibiotic resistance levels were sources and rates of atmospheric mercury accumulation in southern Ontario also lower in the endospore-forming isolates (Table 6). Four recorded by peat cores from three bogs: comparison with natural “back-ground” values (past 8000 years). J Environ Monit 5:935–949.
isolates showed no maximal resistance to any of the clinical Guerra B, Soto SM, Arguelles JM, Mendoza MC. 2001. Multidrug resistance antibiotics tested, and four were maximally resistant to only is mediated by large plasmids carrying a class 1 integron in the emergent one antibiotic. These findings are in support of the co-selection Salmonella enterica serotype [4,5,12:i:-]. Antimicrob Agents Chemother of strains possessing the Hg and antibiotic resistance pheno- type, because both types of resistance were observed to be at Hasebe A, Koike J, Katou H. 2003. Strong retardation in the transport of Burkholderia cepacia during infiltration into a volcanic ash soil. Microbes reduced levels among the sporulating isolates. Additionally, the low abundance of merA positive isolates suggests that Hg was Hobman JL, Essa AM, Brown NL. 2002. Mercury resistance (mer) operons in acting as the selective agent in the bogs and could be inducing enterobacteria. Biochem Soc Trans 30:719–722.
Downloaded By: [Fekete, Frank A.] At: 01:14 7 May 2009 Kilian MR, van der Plicht J, van Geel B. 1995. Dating raised bogs: new aspects of AMS 14C wiggle matching, a reservoir effect and climatic change. QuatSci Rev 14:959–966.
Lane DJ. 1991. Nucleic acid techniques in bacterial systematics. In: Stacke- CONCLUSIONS
brandt E, Goodfellow M, editors. Modern microbiological methods. New This is the first report describing the sphagnum bog environ- ment as a unique natural system in which to study Hg’s influence Lodenius M, Seppanen A, A. U-R. 1983. Sorption and mobilization of mercury on the co-selection of indigenous bacterial strains that possess in peat soil. Chemosphere 12:1575–1581.
Lodenius M, Tulisalo E, Soltanpour-Gargari A. 2003. Exchange of mercury both Hg and multiple drug resistances. merA genes and the between atmosphere and vegetation under contaminated conditions. Sci Total multiple drug resistance phenotype were found in an environ- ment apparently devoid of antibiotics from anthropogenic and Mazel D, Dychinco B, Webb VA, Davies J. 2000. Antibiotic resistance in the natural sources. The multiple antibiotic resistances observed in ECOR collection: integrons and identification of a novel aad gene. Antimi- these indigenous bacterial strains was most likely due to pres- crob Agents Chemother 44:1568–1574.
McArthur JV, Tuckfield RC. 2000. Spatial patterns in antibiotic resistance sure exerted by an indirect selective agent such as a metal. In among stream bacteria: effects of industrial pollution. Appl Environ Mi- the sphagnum bog environment, mercuric ion is sequestered by the sphagnum substrate. This sequestration applies a selective McIntosh D, Cunningham M, Ji B, Fekete FA, Parry EM, Clark SE, Zalinger pressure on bacterial populations, and because of co-resistance ZB, Gilg IC, Danner GR, Johnson KA, Beattie M, Ritchie R. 2008. Transfer- of these genetic determinants, also selects for multiply drug- able, multiple antibiotic and mercury resistance in Atlantic Canadian isolatesof Aeromonas salmonicida subsp. salmonicida is associated with carriage resistant bacteria. Hence, the sphagnum bog environment repre- of an IncA/C plasmid similar to the Salmonella enterica plasmid pSN254.
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