Diversity of Integron- and Culture-Associated Antibiotic Resistance Genes in Freshwater Floc

Abstract

Clinically important antibiotic resistance genes were detected in culturable bacteria and class 1 integron gene cassettes recovered from suspended floc, a significant aquatic repository for microorganisms and trace elements, across freshwater systems variably impacted by anthropogenic activities. Antibiotic resistance gene cassettes in floc total community DNA differed appreciably in number and type from genes detected in bacteria cultured from floc. The number of floc antibiotic resistance gene cassette types detected across sites was positively correlated with total (the sum of Ag, As, Cu, and Pb) trace element concentrations in aqueous solution and in a component of floc readily accessible to bacteria. In particular, concentrations of Cu and Pb in the floc component were positively correlated with floc resistance gene cassette diversity. Collectively, these results identify suspended floc as an important reservoir, distinct from bulk water and bed sediment, for antibiotic resistance in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves and indicate that trace elements, particularly Cu and Pb, are geochemical markers of resistance diversity in this environmental reservoir. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.

INTRODUCTION

Bacterial resistance to antibiotics represents a ubiquitous threat to public health. Although the presence and transmission of antibiotic resistance genes within clinical and agricultural settings are well established, the impact of their continual introduction into natural environments, as well as processes associated with their persistence and ultimate fate, remains unclear. Genes encoding resistance to clinically relevant antibiotics are increasingly being detected in soil and aquatic environments (3, 22, 31), suggesting that environmental bacterial communities represent a significant source of antibiotic resistance elements.

Horizontal gene transfer plays a significant role in the introduction and spread of antibiotic resistance genes within environmental bacterial communities. One pathway for gene transfer is the exchange of integrons and their constituent gene cassettes, each containing a single functional gene, between bacterial cells on conjugative elements. Antibiotic resistance gene cassettes are most commonly associated with class 1 integrons, which are widespread among Gram-negative bacteria (27). In addition to being commonplace in clinical and agricultural isolates, class 1 integrons have been recovered from bacterial communities in a wide range of natural environments (11, 23, 37). The similarity of integrons in environmental bacteria to those found in clinical isolates indicates that antibiotic resistance gene transfer between environmental bacterial communities and bacteria from clinical or agricultural settings can readily occur via these elements (11, 12).

Antibiotic resistance in aquatic bacterial communities is associated with the adaptive acquisition of resistance to trace metals/metalloids (trace elements) in response to trace element-induced stress (28, 36, 38, 39). Genes encoding resistance to antibiotics and trace elements are commonly linked on mobile genetic elements (1, 2, 17, 29), suggesting that trace element exposure can drive the acquisition and persistence of mobile antibiotic resistance genes within bacterial communities. Class 1 integrons are known to be linked with trace element resistance determinants (11, 34) and so may serve as a vehicle for antibiotic resistance gene maintenance by trace element stress.

As both a microhabitat for bacteria and a significant repository for trace elements in aquatic systems (8, 30), suspended floc is likely to be an environmental reservoir for antibiotic resistance genes. Flocs are suspended aggregates of organic and inorganic particles inhabited by highly metabolically active, biofilm-forming microbial communities and typically constitute the principal form of suspended particulate matter in natural aquatic systems (6, 25). As they are highly mobile within aquatic systems and likely exchange bacteria and mobile genetic elements with their surroundings, flocs can potentially contribute to environmental resistance gene transfer. Very little is known about the bacterial constituents of natural aquatic flocs, particularly with respect to their potential role as a reservoir for antibiotic resistance genes. Trace elements (Ag, As, Cu, Co, Ni, and Pb) have recently been shown to be enriched in floc relative to surficial bed sediments in freshwater systems (8, 30). This enrichment may provide sufficient selective pressure for the maintenance of antibiotic resistance genes in floc bacterial communities.

Given the ubiquity of flocs in aquatic environments and their potential to harbor significant quantities of trace elements and bacteria carrying antibiotic resistance genes, the objectives of this study were to (i) establish antibiotic resistance gene occurrence in culturable bacteria and class 1 integron gene cassettes recovered from floc bacterial communities across variably impacted aquatic systems and (ii) identify any links between floc resistance genes and trace element geochemistry.

MATERIALS AND METHODS

Description of study sites.

Four freshwater systems in Ontario, Canada, with varying anthropogenic impacts were selected for comparison: (i) a combined sewer overflow (CSO) outfall in a highly industrialized urban area of Hamilton (43°15′43″N, 79°48′02″W); (ii) an urban public beach (Sunnyside Beach, Lake Ontario) in Toronto impacted by wastewater effluents (43°38′14″N, 79°27′20″W); (iii) a rural stream near Guelph impacted by light agricultural activities (pastureland used to graze livestock) (43°39′24″N, 80°24′06″W); and (iv) a remote lake (Coldspring Lake) accessible only by float plane in a nature preserve area of Algonquin Park (45°85′28″N, 78°82′17″W) (see Fig. S1 in the supplemental material). The Hamilton site was a small, slow-moving, open-water ditch (4 m wide by 0.5 m deep) receiving CSO from the Kenilworth sewershed with a drainage area of 265.5 ha (7). The Guelph site had dimensions similar to those of the CSO ditch and was at base flow during times of sampling. The Toronto and Algonquin Park sites were within the littoral zones of the lakes at total water depths of 1.2 m and 3.5 m, respectively. Sample collection at the four sites was conducted between July and September 2009.

Sampling procedure.

At each site, water and suspended floc samples were collected <0.5 m above the sediment-water interface. All sampling equipment was rinsed in 4% HCl for >24 h and then rinsed eight times with ultrapure water (18.2 MΩ cm; Milli-Q; Millipore) before use. Water samples for trace element analysis were collected, using a Van Dorn sampler, into syringes; sequentially filtered through 0.45- and 0.22-μm syringe filters (Millipore, Billerica, MA); and acidified to 2% (vol/vol) using trace metal grade HNO₃, in triplicate. Samples were stored at 4°C in the dark until analysis. Suspended floc was collected using a continuous-flow centrifuge (model KA 2-06-075; GEA Westfalia Separator, Inc., Northvale, NJ) that pumped water (>2,000 liters at 6 liters min⁻¹) through stainless steel bowls at a rotation speed of 9,470 rpm. The sampling depth was within the euphotic zone for all sites, and floc samples were collected over 6 to 8 h during the day. Floc was transferred from centrifuge bowls into sterile Falcon tubes using sterilized plastic spatulas within 4 h of sampling. Aliquots of the floc were separated for bacterial culturing, with the remainder stored at −20°C for trace element analysis and DNA extraction.

Trace element analysis.

Dissolved aqueous (filtered; <0.22 μm) trace element concentrations and floc trace element concentrations (μmol g⁻¹ [dry weight]) were quantified in triplicate by inductively coupled plasma mass spectrometry (Sciex Elan 6100; Perkin Elmer, Woodbridge, ON, Canada). The specific trace elements analyzed (Ag, As, Cu, and Pb) were selected based on previously identified linkages between genes conferring resistance to these elements and antibiotic resistance genes in mobile genetic elements (35). Field and procedural blanks demonstrated negligible contamination (<5%) for all analyzed trace elements.

As natural flocs, like any aquatic sediment, are heterogeneous multicomponent solids, quantification of total floc trace element concentrations is not sufficient to predict trace element bioavailability. Here, floc trace element analysis followed a sequential extraction technique involving microwave digestion (15), which operationally partitioned floc trace elements associated with five fractions possessing differing trace element reactivities and affinities within the bulk floc: exchangeable (loosely bound), acid-soluble carbonates/sulfides, amorphous Fe/Mn oxyhydroxides, crystalline Fe/Mn oxides, and organics/sulfides. Sequential extractions selectively dissolved each floc fraction, concomitantly releasing any trace elements associated with that matrix fraction into the supernatant, which were then quantified. This approach provides a means to determine both whole-floc trace element concentrations and the partitioning of trace elements among the differentially reactive and remobilizable floc solid fractions. Thus, sequential extractions provide greater insight into constituent pools of floc accessible to bacteria and may indicate relevant trace element pools linked to antibiotic resistance.

Culturing floc bacteria.

Each floc sample was resuspended in 1× phosphate-buffered saline (PBS) with 0.625% (vol/vol) Tween 80, and serial dilutions were spread onto plates of yeast extract agar (a general nutrient-rich medium for heterotrophic Gram-positive and Gram-negative bacteria) and Sorbitol-MacConkey agar (a selective nutrient-rich medium for heterotrophic Gram-negative bacteria). Cultures were incubated aerobically at 37°C for 48 h.

DNA extraction.

For each plate culture, the entire medium surface was scraped to collect all bacterial colonies for DNA extraction. This approach potentially captures bacterial taxa that cannot be isolated on solid media owing to their dependence on growth factors from other bacteria (5). Genomic DNA was extracted from pooled colonies using the DNeasy Blood and Tissue Kit (Qiagen, Mississauga, ON, Canada). Total community DNA was extracted from floc samples using the PowerSoil DNA Isolation Kit (MO Bio Laboratories, Carlsbad, CA) according to the manufacturer's instructions.

Recovery of gene cassettes.

Gene cassettes present in class 1 integrons were selectively amplified from floc total community DNA by PCR using HS286/HS287 primers targeting recombination sites that delimit individual cassettes (37). The PCR products were separated by gel electrophoresis and purified using the MinElute Gel Extraction Kit (Qiagen, Mississauga, ON, Canada).

Microarray-based detection of antibiotic resistance genes.

Pooled genomic DNA from colonies scraped from plates of medium for each floc sample and gel purified class 1 integron gene cassette PCR products were investigated for the presence of 54 antibiotic resistance gene families associated with Gram-negative bacteria using the Identibac AMR-ve miniaturized DNA microarray-based assay (Identibac, New Haw, Surrey, United Kingdom). The assay was carried out according to the manufacturer's instructions. Individual genes or sets of related gene variants corresponding to individual microarray spots were interpreted as being present in a sample when a relative signal of at least 0.05 was recorded for each of three corresponding spots.

To confirm the microarray results, resistance genes covering several antibiotic classes were targeted for PCR amplification from pooled cultured bacterial genomic DNA and gene cassette PCR products. Primers and PCR conditions specific for aac(3)Ia (20), aadA1 (20), bla_ACT-1 (designed for this study; 5′-CTTTGCTGCGCCCTGCTGCTC-3′ and 5′-TAAAKGCCACGTAGCTGCCAAACC-3′; annealing temperature, 63°C), bla_LEN-1 (16), sul2 (26), tetA (13), and tetC (10) genes were employed for this task. The PCR products were separated by gel electrophoresis. If present, a clean band corresponding to the expected PCR product size was excised from gels and then purified and sequenced as described previously. Smeared bands were excised, gel purified, and cloned using the ZeroBlunt Topo PCR Cloning Kit (Invitrogen, Carlsbad, CA), and several clones containing inserts were sequenced. The closest matches to the sequences were determined by searching the NCBI nucleotide database with BLAST.

Sequencing of clones generated from PCR amplification with primers targeting individual antibiotic resistance genes largely confirmed the results of the microarray assay (Table 1). PCR products had high (99 to 100%) sequence homology with expected target genes [aac(3)Ia, aadA1, sul2, tetA, tetC] in the GenBank nucleotide database (as of March 2012) with one exception. The closest match for two distinct sequences recovered from culturable Coldspring L. floc bacteria using primers intended to target bla_ACT-1, an Ambler class C beta-lactamase, was a beta-lactamase gene of the same class with only 76% homology to the recovered sequences.

Table 1.

Antibiotic resistance genes detected in cultured bacteria and class 1 integron gene cassettes recovered from suspended floc across four variably impacted freshwater systems

Resistance	Gene(s)	Detectiona
		Hamilton		Toronto		Guelph		Coldspring		Total
		C	I	C	I	C	I	C	I	C	I
Aminoglycoside transferase	aac(3)Ia		+		+		+			3	10
	aac(3)IVa				+
	aac(6′)Ib		+
	aadA1 like	+	+
	aadA2 like		+
	aadA4 like		+		+
	ant(2″)Ia		+
	strB	+				+
Beta-lactamase (class A)b	blaCTX-M-1		+		+					3	5
	blaCTX-M-9		+
	blaLEN-1								+
	blaSHV	+
	blaTEM-1	+	+					+
Beta-lactamase (class D)	blaOXA-2		+	+	+					1	5
	blaOXA-7		+		+
	blaOXA-9				+
Beta-lactamase (class C)	blaACC-1				+					10	5
	blaACC-2							+
	blaACT-1	+	+	+		+		+
	blaCMY	+		+				+
	blaFOX			+
	blaMOX		+		+				+
	blaMOX-CMY							+
Chloramphenicol efflux pump	cmlA1 like		+
Chloramphenicol acetyltransferase	catB8		+
Erythromycin esterase	ereB		+	+	+
DNA gyrase protection protein (quinolone)	qnrB				+
	qnrS			+
Modified dihydropteroate synthase (sulfonamide)	sul1	+
	sul2		+						+
Streptogramin acetyltransferase	vatE		+
Tetracycline efflux pump	tetA	+								4	2
	tetC		+
	tetD			+
	tetE	+		+			+
Modified dihydrofolate reductase (trimethoprim)	dfr12			+						3	4
	dfrA1	+
	dfrA7		+				+
	dfrA15			+
	dfrA17		+				+
Total		10	21	10	11	2	4	5	3

^aC, culture-based detection method (combined results for pooled isolates from each type of culture medium); I, integron-based detection method; +, gene or group of gene variants was detected. The shading indicates detection of a gene or related sequence by single-gene PCR and sequencing.

^bBeta-lactamase class follows the Ambler classification system.

Nucleotide sequence accession numbers.

The beta-lactamase gene sequences recovered from culturable Coldspring L. floc bacteria were deposited in the GenBank nucleotide sequence database (GenBank/EMBL/DDBJ) under accession numbers JQ790529 and JQ790530.

RESULTS

Antibiotic resistance genes in suspended floc.

Genes encoding resistance to clinically relevant antibiotics were detected in both bacteria cultured from floc and class 1 integron gene cassettes recovered from floc total community DNA across all four sites investigated in this study (Table 1). The diversity of antibiotic resistance genes varied across sites and between cultured bacteria and gene cassettes. Hamilton floc and Toronto floc (urban sites) ranked first and second, respectively, in the number of resistance gene types (cultured plus gene cassette), antibiotic classes represented by resistance gene types (cultured plus gene cassette), and gene cassette resistance gene types across sites. Fewer gene types were detected in Guelph (agricultural site) floc than in Coldspring L. (a remote Algonquin Park site) floc, but Coldspring L. gene types represented fewer antibiotic classes. Coldspring L. genes were predominantly Ambler class C beta-lactamase genes detected in cultured bacteria.

There was little overlap between antibiotic resistance gene types detected in cultured bacteria and gene cassettes across all sites, with a greater number of gene types detected in gene cassettes. Also, the distributions of gene types between cultured bacteria and gene cassettes across all sites varied according to the gene family. Genes encoding aminoglycoside-modifying enzymes and Ambler class A and D beta-lactamases were mostly present as gene cassettes, while class C beta-lactamase genes and tetracycline resistance genes were predominantly found in cultured bacteria.

Across sites, the number of gene cassette antibiotic resistance gene types and antibiotic classes represented by gene types in floc were in agreement with the expected degree of anthropogenic impact. Genes encoding resistance to chloramphenicol, erythromycin, streptogramins, and quinolones were detected only in flocs from the more heavily impacted Hamilton and Toronto sites.

Suspended floc trace element geochemistry.

Total aqueous (dissolved; <0.22 μm) trace elements at the depth of floc collection and total floc trace element concentrations (both reported as the sum of Ag, As, Cu, and Pb) varied appreciably across sites (Table 2). Concentrations of total aqueous trace elements were highest at the Hamilton site (0.182 μmol liter⁻¹), an order of magnitude higher than at the other sites, and lowest at the Guelph site (0.019 μmol liter⁻¹) and the Coldspring L. site (0.020 μmol liter⁻¹). Similarly, total floc trace element concentrations were highest at the Hamilton site (2.11 μmol g⁻¹) and lowest at the Guelph site (0.53 μmol g⁻¹). The concentrations of individual trace elements (Ag, As, Cu, and Pb) in each floc fraction are provided in Table S1 in the supplemental material.

Table 2.

Trace element concentrations in water at floc collection depth and in suspended floc fractions accessible to bacteria

Parameter	Concnc
	Hamilton	Toronto	Guelph	Coldspring
Aqueousa total TEb (μmol liter−1)	0.182	0.043	0.019	0.020
Floc total TE associated with exchangeable fraction (μmol g−1)	0.05	0.06	0.01	0.00
Floc total TE associated with acid-soluble carbonate/sulfide fraction (μmol g−1)	0.39	0.13	0.01	0.01
Floc total TE associated with amorphous Fe/Mn oxyhydroxide fraction (μmol g−1)	0.52	0.25	0.10	0.65
Floc total TE associated with crystalline Fe/Mn oxide fraction (μmol g−1)	0.93	0.26	0.16	0.28
Floc total TE associated with organic/sulfide fraction (μmol g−1)	0.21	0.38	0.24	0.66

^a0.22-μm filtered.

^bTE, trace elements Ag, As, Cu, and Pb.

^cTrace element concentrations are mean concentrations of three replicate samples. Standard deviations were on the order of 10⁻³ μmol liter⁻¹ for aqueous-element concentrations and 10⁻² μmol g⁻¹ for floc element concentrations.

Suspended floc trace elements and antibiotic resistance gene diversity.

The total number of antibiotic resistance gene cassette types detected in floc was positively correlated with both total (the sum of Ag, As, Cu, and Pb) aqueous trace element concentrations (r = 0.95; n = 4; P < 0.05) at the floc collection depth and total trace element concentrations collectively associated with the exchangeable and acid-soluble carbonate/sulfide floc fractions (r = 1.00; n = 4; P < 0.01). Of all elements examined, only the concentrations of Cu (r = 0.98; n = 4; P < 0.01) and Pb (r = 0.99; n = 4; P < 0.01) collectively associated with the exchangeable and acid-soluble carbonate/sulfide fractions were strongly positively correlated with the number of antibiotic resistance gene cassette types.

DISCUSSION

The detection of antibiotic resistance genes in floc culturable bacteria and total community DNA across highly variable sites identifies suspended floc as a reservoir for resistance, even in remote locations not appreciably impacted by anthropogenic activities. Further, the breadth of antibiotic resistance gene types detected in floc indicates that sampling only bulk water (i.e., unconcentrated floc) and/or bed sediments is insufficient to assess resistance gene diversity in aquatic systems. The minimal overlap and similar numbers of gene types detected in floc cultured bacteria and gene cassettes across sites suggests that nonculturable bacteria constitute a distinct reservoir for antibiotic resistance genes in natural systems. The observation of antibiotic- and beta-lactamase class-specific resistance gene distributions in gene cassettes and cultured bacteria indicates that certain groups of related gene types may be more strongly associated with class 1 integrons and/or nonculturable bacteria. In particular, the predominantly chromosomal class C beta-lactamases were the only beta-lactamase class found predominantly in cultured bacteria versus within gene cassettes in floc across sites. Beta-lactamases are ancient enzymes originally encoded in bacterial chromosomes, and those belonging to class C have been mobilized to a substantial degree on plasmids only in the last few decades in clinical settings (21). In agreement with previously characterized class 1 integron gene cassettes from wastewater and freshwater systems (4, 24, 33, 41), genes encoding aminoglycoside-modifying enzymes, dihydrofolate reductases, class D beta-lactamases, and chloramphenicol-modifying enzymes were present as gene cassettes in floc. The additional detection of gene cassettes containing genes encoding class A and C beta-lactamases and resistance to quinolones, sulfonamides, streptogramins, and tetracyclines in this study indicates that antibiotic gene cassette diversity associated with class 1 integrons is greater than previously thought. Given the horizontal mobility of integrons, these results provide evidence that a wide variety of clinically important antibiotic resistance genes are mobile within aquatic bacterial communities, particularly those associated with floc. The presence of a tetC gene cassette in Coldspring floc was not expected. Tetracycline resistance genes have not been previously found as gene cassettes (32). Further, Coldspring L. is situated within a nature reserve accessible only by float plane, suggesting the cassette may be endemic to bacteria found within the lake and more broadly within environmental aquatic systems. The sequences of a class C beta-lactamase gene identified by the microarray assay as bla_ACT-1 in bacteria cultured from Coldspring floc did not closely match any known clinical antibiotic resistance genes, indicating that it is a novel gene that is also endemic to the site.

The positive association between floc antibiotic resistance gene diversity and both aqueous and floc exchangeable and acid-soluble carbonate/sulfide-associated trace element concentrations indicates that trace elements, particularly Cu and Pb in floc, are a marker of diversity in this environmental reservoir. In general, aqueous trace elements are more bioavailable to suspended floc bacteria than those within bed sedimentary compartments, as the porous nature of flocs permits exposure of inhabitant bacteria to contaminants present in the surrounding bulk water (6). Trace elements associated with the exchangeable floc fraction are loosely bound and so are readily accessible to floc bacteria. The acid-soluble fraction of floc is easily dissolved with environmentally relevant decreases in pH and thus would release any trace elements with increasing acidity. Further, iron(II) sulfides are accessible to iron- and sulfur-oxidizing bacteria as electron sources, and thus, their metabolic activity could also result in the release of trace elements, which would then be readily accessible to various functional guilds of bacteria associated with floc. The positive associations seem logical, with acquisition and maintenance of antibiotic resistance genes in natural bacterial communities possibly resulting from coselection of antibiotic and trace element resistance genes linked on mobile genetic elements driven by trace element exposure. However, not all trace elements are equally bioavailable or toxic (9), and element specificity likely also plays a role. Little is known about associations between resistance genes for these metals and integrons or antibiotic resistance genes. A Pb resistance gene cluster was found alongside genes encoding resistance to all of the major classes of antibiotics detected in this study in transferable plasmids from clinical Enterobacter isolates (2), so Pb may be implicated in determining the diversity of antibiotic resistance genes associated with mobile genetic elements in floc. Similarly, genes conferring resistance to Cu and clinically relevant antibiotics have been found on a transferable plasmid from an agricultural isolate (19). Class 1 integron abundance has been directly correlated with the degree of anthropogenic impact in aquatic bacterial communities, indicating that gene transfer is enhanced in communities experiencing trace element stress (18, 34, 40). Rosewarne et al. (34) identified a strong positive relationship between class 1 integron abundance and levels of Cu, Hg, Pb, and Zn in freshwater benthic bacterial communities. Expression of the integrase gene, which dictates the magnitude of gene cassette mobility, is increased following activation of the SOS response, a general bacterial response to environmental stressors (14). As a result, the presence of trace elements at stressing concentrations is expected to stimulate gene cassette exchange by integrons in bacterial communities as an adaptive response, providing a putative explanation for the positive association between antibiotic resistance gene cassette diversity and trace element concentrations in floc.

Coupling the recovery of gene cassettes by PCR with a DNA microarray-based assay for the detection of known antibiotic resistance genes offers a culture-independent means of accessing mobile resistance genes within natural bacterial communities. The detection of class C beta-lactamase gene sequences in Coldspring floc with relatively low homology to known genes of this class suggests that the microarray-based approach can access endemic antibiotic resistance genes in natural bacterial communities. As such, it is a useful tool for identifying genes related to clinically important resistance genes for further investigation. A caveat to this approach is that although PCR amplification of gene cassettes from total community DNA permits access to integrons in nonculturable bacteria, only a fraction of environmental gene cassettes are recovered because primer design is based solely on known integron sequences, and thus, primers will preferentially bind to and amplify certain templates. In several cases, disparities were observed between antibiotic resistance gene types detected using the microarray assay and by single-gene PCR. These disparities may reflect differences in the primer and/or probe sequences used for each approach. Further, the microarray assay utilized a two-step method (PCR plus DNA microarray) that likely increased its sensitivity relative to the visualization of single-gene PCR products by gel electrophoresis.

In conclusion, suspended floc has been identified as an important and distinct reservoir for antibiotic resistance genes in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves. Across systems, trace elements, particularly Cu and Pb, are geochemical markers of antibiotic resistance diversity in floc. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.