Real Time PCR for the Rapid Detection of

Environ. Sci. Technol. 2009, 43, 3343–3348

Real Time PCR for the Rapid Detection of vanA Gene in Surface Waters and Aquatic Macrophyte by Molecular Beacon Probe PUSHPA LATA,† SIYA RAM,† MADHOOLIKA AGRAWAL,‡ AND R I S H I S H A N K E R * ,† Indian Institute of Toxicology Research (CSIR), P.O. Box 80, Mahatma Gandhi Marg, Lucknow-226001, U.P., India, and Banaras Hindu University, Varanasi-221005, U.P., India

Received December 22, 2008. Revised manuscript received March 9, 2009. Accepted March 12, 2009.

Enterococci serve as an “indicator” of fecal contamination for recreational water quality. The vancomycin-resistantenterococci (VRE) are emerging environmental contaminants in the surface waters. The aim of this study was to develop a rapid and specific molecular beacon probe (MBP)-based real-time PCR assay for detection of vanA gene in surface waters and aquatic macrophyte. The limit of detection (LOD) of the MBP assay was 1 CFU/mL of VRE [r ) 0.943; PCR efficiency ) 99.7%] in 2-fold dilution format within 2.5 h and demonstrated high specificity for environmental enterococci isolates exhibiting VanA phenotype (n ) 25). VRE were detected from downstream surface waters of the rivers impacted by point sources of pollution and recreational activities. The probe detected vanA gene in rootmat associated microbiota of E. crassipes (Mart.) Solms. an aquatic nuisance weed, at eutrophic sites of the surface waters (ANOVA p < 0.001). In addition, the assay enabled detection of otherwise nondetectable vanA gene concentration in the upstream sites of two Indian rivers (Student’s t test p < 0.001). The MBP assay developed can be used for sensitive and rapid detection of VRE in surface waters and identification of nonpoint sources of pollution for implementation of preventive measures to protect human health.

Introduction Enterococci are among the common microbiota of the gastrointestinal tract of humans and animals. Regulatory guidelines recommend enterococci as an indicator of fecal contamination in management of recreational water quality as their presence correlates best with the incidence of swimming-related gastroenteritis (1, 2). The members of genus enterococcus are also opportunistic pathogenic bacteria and have emerged as major cause of nosocomial infections worldwide (3-9). Two species responsible for the majority of human infections are Enterococcus faecalis and Enterococcus faecium (10, 11). The main reason for survival of these organisms in the environment is their intrinsic resistance to several commonly used antimicrobials and * Corresponding author phone: 91+ 0522 -2613786/2614118/ 2627586extn.237; fax: 91+ 0522-2611547; e-mail: [email protected], [email protected], [email protected]. † Indian Institute of Toxicology Research (CSIR). ‡ Banaras Hindu University. 10.1021/es803635y CCC: $40.75

Published on Web 04/02/2009

 2009 American Chemical Society

ability to acquire resistance against all currently available antimicrobials (e.g., vancomycin) either by mutation or transfer of plasmids and transposons (12, 13). Vancomycin, an antimicrobial of the glycopeptide group, once considered as the bastion in the treatment of enterococcal infections is no longer effective in managing antimicrobial-resistant enterococci. Vancomycin-Resistant Enterococci (VRE), first described in 1988, are now ubiquitous among clinical, food, and surface water samples (14-18). The prevalence of antimicrobial-resistant and pathogenic indicator bacteria (viz. VRE, shiga toxin, and enterotoxin producing E. coli) in surface waters is a major concern for public health in India as a large population depends on unprocessed water for various daily needs (19). Vancomycin resistance has been classified into different groups based on phenotypes including VanA, VanB, and VanC (20). The VanA phenotype possesses inducible, high-level resistance to vancomycin (MICs, g 64 mg/mL) and teicoplanin (MICs, g16 mg/mL). VanA type of resistance occurs in the presence of vanA gene cluster reported on transposon Tn1546 (21). VanA protein is a ligase of altered substrate specificity which produces altered cell wall precursor D-AlaD-Lac in preference to actual cell wall precursor and the target of vancomycin D-Ala-D-Ala (22). Many reports are available in the literature regarding the identification of VRE using conventional microbiological methods, which require time, resources, and space (23-26). The application of conventional polymerase chain reaction technique for detection of VRE, although an improvement over conventional microbiological or biochemical tests, lacks absolute quantitation and requires time-consuming post PCR analysis (23, 27). However there is no report on rapid, cultureindependent detection of VRE and viable but nonculturable (VBNC) entities from surface waters. Real-time PCR has been used to quantify the presence of microorganisms in complex environmental matrices (28, 29). This method improves the precision and sensitivity of conventional PCR by addition of a fluorescently labeled probe so that the target gene can be detected and quantified without post-reaction analysis. This study envisages a molecular beacon probe (MBP)based real-time PCR assay for rapid and culture-independent detection of vanA gene. The objectives of the current study were (i) to design, test, and enhance the sensitivity of a MBPbased quantitative PCR assay for the detection of vanA gene in Enterococcus spp.; and (ii) to validate the specificity and efficacy of the assay for detection of vanA gene from (a) surface water samples of rivers Ganga and Gomti; (b) rootmats of Eicchornia crassipes (Mart.) Solms, an aquatic nuisance weed which flourishes in eutrophic fresh water bodies collected from respective up- and downstream locations on river Gomti; and (c) environmental enterococci isolates exhibiting VanA, VanB, and vancomycin susceptible phenotypes characterized earlier in our laboratory.

Materials and Methods Bacterial Strains and Culture Conditions. Five different Enterococcus spp., viz. E. faecium ATCC 51559 (vanA positive control), E. faecium ATCC 27270, ATCC 35667 (vanA negative control), E. faecalis ATCC 51299 (vanB positive control), E. durans ATCC 19432, E. hirae ATCC 9790, and E. avium ATCC 14025 procured from American type Culture Collection (Manassas, VA) were used as reference strains. Environmental isolates of enterococci from various surface waters representing VanA (n ) 25), VanB (n ) 33), and vancomycin susceptible (n ) 20) phenotypes were used for testing specificity by the real-time PCR assay. VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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In brief, all reference strains of different Enterococcus spp. were grown in Brain Heart Infusion (BHI) broth at 37 ( 1 °C on a rotary shaker (Innova 4230 New Brunswick, USA) at 150 rpm to approximate optical density of 1.0 at 600 nm. To carry out a background simulation experiment, five different Enterococcus spp. and two non-vanA reference strains of E. faecium were spiked in 1:1 ratio. Further, two sets of cultures were diluted for 10-fold and 2-fold dilution series; one set used for standard plate count method to calculate CFU/mL (Colony Forming Units) and another for DNA extraction. In the data presented throughout this manuscript 1 CFU represents 1 genomic equivalent of VRE exhibiting vanA gene. All experiments were performed in triplicate. Real-Time PCR Assay and Standard Curve Generation. To establish a quantitative PCR for detection of vanA gene, a standard curve was constructed with serial 10-fold and 2-fold dilutions of Enterococcus faecium ATCC 51559 as vanA positive control. In brief, for preparation of standard curve Enterococcus faecium ATCC 51559 grown at approximately 1 O.D. at 600 nm was 10-fold serially diluted in sterile physiological saline to yield 106 to 1 CFU/mL; 2-fold serial dilution performed to yield 104 to 1 CFU/mL as estimated by standard plate count method. DNA template prepared from each dilution was used for standard curve generation (see Supporting Information). MB probe and primer sequences designed in silico for this study are listed in Table S1. The MBP (Vn‘P’: TET-5′-CGCGATCTGTGAGGTCGGTTGTGCGGTATTGATCGCG-3′-BHQ1) and primers (VnA‘F’: 5′-ATCGGCAAGACAATATGACAGC-3′ and VnA ‘R’: 5′-AGCCTGATTTGGTCCACCTC-3′) were synthesized by Metabion (Gmbh, Germany). Briefly, each 50 µL reaction contained dNTPs (0.2 mM), Hotstart Taq DNA polymerase (1 unit, MBI Fermentas, USA) and Taq buffer (1X), MgCl2 (3.0 mM), primers (0.3 µM, each), MB probe (0.3 µM), and 5 µL of DNA template (approximately 3ng-3fg DNA). The PCR protocol was as follows: initial denaturation at 95 °C for 4 min and then 40 cycles at 95 °C for 15 s, 62.3 °C for 10 s, and 72 °C for 15 s. Real-time PCR assays were performed using iCycler real-time PCR system (BIO-RAD, USA) and the standard curve was generated by the iCycler iQ Optical System Software Version 3.0a. The negative template control (NTC) consisted of the PCR reagent master-mix and 5 µL of sterile molecular grade water as a substitute of DNA template. All the realtime PCR assays were performed in triplicate. Sensitivity and Specificity of the Real-time PCR Assay. Five different experiments were conducted to enhance, evaluate, and establish the LOD and specificity of the MBP based real-time PCR assay designed for detection of Enterococcus spp. specific vanA gene. (a) To enhance the LOD of PCR assay in the presence of same background, all aforementioned standard replicates with serially 2-fold diluted E. faecium ATCC 51559 (vanA positive control) were spiked with an additional 200 CFU/mL of E. faecium ATCC 51559. In brief, E. faecium ATCC 51559 grown to 1 O.D. at 600 nm and 200 CFU/mL of this strain was spiked in all serially 2-fold diluted cultures of E. faecium ATCC 51559 from 104 to theoretically 10-1 CFU/mL. (b) To evaluate the LOD in the presence of background of different non-vanA E. faecium strains, two vanA negative E. faecium reference strains ATCC 27270 and 35667 were used to generate a simulated background. In brief, vanA positive E. faecium ATCC 51559, vanA negative E. faecium ATCC 27270 and ATCC 35667 reference strains were grown to 1 O.D. at 600 nm and spiked in 1:1 ratio (i.e., 104 CFU/mL of E. faecium ATCC 51559 spiked with 104 CFU/mL of each E. faecium ATCC 27270 and ATCC 35667) in all serially 2-fold diluted replicates (spiked with additional 200 CFU/mL) of E. faecium ATCC 51559 from 104 to 1 CFU/mL. (c) To evaluate the LOD of MBP based real-time PCR assay for the detection of vanA gene in E. faecium ATCC 51559 (vanA positive control) in the 3344

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presence of background of different enterococci, five Enterococcus spp. (E. faecium ATCC 27270; vanA negative, E. faecalis ATCC 51299; vanB positive, E. durans ATCC 19432, E. hirae ATCC 9790, and E. avium ATCC 14025) were used to generate an intense synthetic multigenomic background. In brief, all five reference strains were grown at approximately 1 O.D. at 600 nm and spiked in 1:1 ratio in all serially 2-fold diluted replicates spiked with additional 200 CFU/mL of E. faecium ATCC 51559 from 104 to 1 CFU/mL as described earlier. (d) To establish the MBP-based real-time PCR assay format for detection of vanA gene from the surface waters and from E. crassipes root-mats using culture-independent methods. In brief, 2.5 L of different surface water samples were centrifuged at 20,000g, 4 °C for 15 min (model CR22G, Hitachi Koki Co. Ltd., Japan). The root-mat-associated microbiota was recovered with some modification in the previous protocols (30, 31). Succinctly, Eicchornia crassipes root-mats (30 ( 0.5 g fresh weight) were sonicated for 30 s in 100 mL of phosphate-buffered saline and then kept on a rotary shaker for 10 min to release the root-mat-associated microbiota. Subsequently root-mats were rinsed thrice in 50 mL of phosphate-buffered saline, then centrifuged at 1,500g for 5 min. to recover microbiota. The sonication of root-mat was carried out thrice at amplitude of 30% and pulse time of 0.5 s with 30 s interval at 4 °C (UP200S Ultrasonic processor, dr. hielscher GmbH, Germany). This was followed by three sequential rinsings to provide an average recovery of 95% of the bacteria from the root-mat surface. DNA was extracted from all samples (see Materials and Methods, Supporting Information). For LOD enhancement, the upstream river water samples were spiked with 200 CFU/mL of E. faecium ATCC 51559. (e) The specificity of the PCR assay was validated by a panel of VanA (n ) 25), VanB (n ) 33), and vancomycin susceptible (n ) 20) phenotypes of enterococci (Table S2). Phenotypic characterization of VRE and non-VRE environmental isolates was performed using vancomycin (Va: 30 µg/disk) and teicoplanin (Te: 30 µg/disk) by the Kirby-Bauer disk diffusion method as described by CLSI (32). In brief, pure culture colonies (3-4) were transferred to tubes containing 5 mL of BHI broth and incubated in a rotary shaker (INNOVA 4230, New Brunswick, USA) at 150 rpm and 37 ( 1 °C for 4-6 h to yield a suspension of 106 cells/mL. The inoculum was uniformally spread on sterile Mueller-Hinton agar plates (90 mm diameter) using a sterile cotton swab. Four to six antimicrobial discs were applied aseptically, at least 24 mm apart on Mueller-Hinton agar plates. The plates were incubated immediately at 35 ( 1 °C; 16-18 h for teicoplanin and 24 h for vancomycin. The test was performed in triplicate for each enterococcus isolate. The diameters of zones showing inhibition were measured to the nearest mm and recorded. A zone size interpretive chart was used to determine sensitivity/resistance of antimicrobials as described by CLSI (2005). Genotypic characterization of these isolates was performed using housekeeping gene tuf; specific for genus enterococcus and variants of gene sodA specific for E. faecalis and E. faecium as described elsewhere (33, 34). Statistical Analyses. The equation of line and correlation coefficient (r) were simultaneously calculated by iCycler iQ Optical System Software Version 3.0a. The real-time PCR amplification efficiency (E) was computed using slope of the standard curve through the equation E ) [10(-1/slope)] - 1. A slope of -3.322 represents an efficiency of 100%. Slopes of the standard curves, correlation coefficients, and PCR amplification efficiencies of different experiments were compared for estimating LOD. The significance of difference in LOD values of direct and enhancement experiments and VRE contamination level in surface water and root-mats of E. crassipes at two sites on river Gomti were compared using one way analysis of variance (ANOVA) with the posthoc Tukey’s multiple comparison test. The load of VRE con-

FIGURE 1. Standard curve generated from 10-fold serial dilution (106 to 101 CFU/mL) of E. faecium ATCC 51559 grown in BHI broth for 12 h at 37 ( 1 °C to approximately 1 O.D. at 600 nm. (1 CFU represents 1 genomic equivalent of VRE exhibiting vanA gene).

TABLE 1. Comparison of Direct 2-Fold Experiment with Spiked 2-Fold Experiments a, b and ca Conducted to Enhance the LOD of vanA Gene Molecular Beacon Probe-Based Real-Time PCR Assay spiked experiment (2-fold) type of experiment LOD ( SD significance correlation coefficient (r) linear regression PCR efficiency (%) mode of detection

2-fold 20 ( 1.00 0.980 Y ) -3.408X + 38.891 96.5 direct

a

b

c

1 ( 0.28 Rb 0.943 Y ) -3.328X + 37.544 99.7 LOD enhancement

5 ( 0.45 Rbβb 0.879 Y ) -3.345X + 37.911 99.0 LOD enhancement

5 ( 0.50 Rbβb 0.940 Y ) -3.422X + 37.769 96.0 LOD enhancement

a Experiment a: spiked with 200 CFU/mL of E. faecium ATCC 51559; b: spiked with two non-VRE E. faecium ATCC strains (1:1) + 200 CFU/mL of E. faecium ATCC 51559; c: spiked with five ATCC strains of different Enterococcus spp. (1:1) + 200 CFU/mL of E. faecium ATCC 51559. b p < 0.001 (One way ANOVA with the posthoc Tukey’s multiple comparison test); R: difference between direct 2-fold and spiked 2-fold experiments; β: difference between spiked 2-fold a, b, and c experiments respectively.

tamination at two sites in river Ganga surface waters was compared using Student’s t test. Significance of all statistical tests was set at P < 0.05. One way ANOVA and Student’s t test were performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, www.graphpad. com).

Results Sensitivity and Specificity of the Real-time PCR Assay. To establish a quantitative PCR for detection of VRE exhibiting vanA gene, a standard curve of serial 10-fold dilutions (from 106 down to 1 CFU/mL) and serial 2-fold dilutions (from 104 to 1 CFU/mL) of positive control (E. faecium ATCC 51559) was constructed (Figure 1; Figure S1). Furthermore, no detectable amplification occurred in the negative template control. The lowest detection limit of the quantitative realtime PCR using MBP was 102 CFU/mL (r ) 0.991; PCR efficiency E ) 98.8%) and 20 CFU/mL (r ) 0.980; E ) 96.5%) in 10 and 2-fold dilution format respectively (Figures S1, S5, S6). Further, the outcomes of five different experiments conducted to enhance, evaluate, and establish the sensitivity limit or LOD and specificity of the MBP-based real-time PCR assay are as follows: (a) The LOD of the quantitative PCR assay was enhanced to 1 CFU/mL (r ) 0.943; E ) 99.7%) with spiked 200 CFU/mL of vanA positive control in its 2-fold dilution replicates (Figure S2, S7). (b) The LOD of real-time

PCR assay was 5 CFU/mL in the presence of the simulated background of two different non-vanA E. faecium ATCC reference strains (r ) 0.879; E ) 99.0%; Table 1; Figure S3). (c) The LOD of the assay was 5 CFU/mL in the presence of an intensely synthetic multigenomic background comprising five Enterococcus spp. reference strains from ATCC viz. E. faecalis, E. faecium, E. durans, E. avium, and E. hirae (r ) 0.940; E ) 96.0%; Table 1; Figure S4). (d) Culture-independent quantitative detection of vanA gene directly from surface waters samples and microbiota recovered from Eicchornia crassipes root-mat through MBP based real-time PCR assay was established in this study. The MBP could detect vanA gene quantitatively (r ) 0.861; E ) 96.7%) in surface waters from the upstream and downstream sites on river Ganga in Kanpur city and also Eicchornia crassipes plant samples from respective sites on river Gomti in Lucknow city (Tables 2 and 3). The maximum concentration of the vanA gene was recorded from the root-mat of E. crassipes collected at Gandhi setu on river Gomti followed by river Ganga surface water at Jajmau, Gandhi setu surface water, and root-mat of E. crassipes from Gaughat (Table 2). The upstream surface water samples of both rivers were observed to have nondetectable concentration of vanA gene (Tables 2 and 3). Further enhancement of LOD was recorded by spiking 200 CFU/mL of E. faecium ATCC 51559 into these upstream surface water samples (Tables 2 and 3). The vanA gene concentration was 548-fold higher in the E. crassipes root-mat-associated VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Culture-Independent Quantitative Detection of vanA Gene by MBP-Based Real-Time PCR Assay in Surface Water and E. crassipes Root-Mat-Associated Multigenomic Samples from Eutrophic River Gomti along Rural-Urban Setting of Lucknow City Impacted by Untreated Sewage from Surrounding Ecozones River Gomti sampling site sample type

Gau-ghat (upstream) surface water

CFU/100 mL ( SD significance correlation coefficient (r) linear regression PCR Efficiency (%) mode of detection

4 ( 0.03 0.748 Y ) -3.383X + 36.169 97.5 LOD enhancement

Gandhi setu (downstream)

E. crassipes root-mat-associated

surface water

E. crassipes root-matassociated

2.11 × 103 ( 5.77 Ra 0.861 Y ) -3.403X + 38.132 96.7 direct

4.46 × 102 ( 0.57 βa 0.861 Y ) -3.403X + 38.132 96.7 direct

1.68 × 104 ( 104.10 Ra βa 0.861 Y ) -3.403X + 38.132 96.7 direct

a p < 0.001 (One way ANOVA with the posthoc Tukey’s multiple comparison test); R: difference in VRE concentration between surface water and E. crassipes root-mat-associated microbiota; β: difference in VRE concentration between upand downstream sites.

TABLE 3. Culture-Independent Quantitative Detection of vanA Gene by MBP-Based Real-Time PCR assay in Surface Water Multigenomic Sample of River Ganga along Rural-Urban Setting of Kanpur City Impacted by Untreated Sewage from Surrounding Ecozones River Ganga sampling site sample type CFU/100 mL ( SD significance correlation coefficient (r) linear regression PCR efficiency (%) mode of detection

Bithoor (upstream site) Jajmau (downstream) surface water surface water 5 ( 0.02

6.42 × 103 ( 5.29 Ra

0.748

0.861

Y ) -3.383X + 36.169 Y ) -3.403X + 38.132 97.5 96.7 LOD enhancement direct

a p < 0.001 (Student’s t test); R: difference in VRE concentration between up and downstream sites.

microbiota than surface water from upstream site and the burden of vanA gene contamination in downstream surface water was 115-fold greater compared to upstream site on river Gomti. On the other hand the downstream site on river Ganga when compared to upstream site was observed 1100fold more contaminated with vanA gene. All the sites varied significantly in terms of vanA gene contamination (ANOVA p < 0.001). (e) The specificity of the PCR assay was validated using 4-fold (1 × 102 CFU/mL) diluted enterococci cultures. The amplification signals identical to vanA positive control were obtained with the environmental enterococci isolates showing VanA phenotype but no amplification signals were observed in VanB phenotype and vancomycin susceptible isolates (Table S2).

Discussion In the past decade, VRE have emerged as important human pathogens implicated in severe complications including fatal sepsis and multiple-organ failure (3-9, 35). There are several reports on swimming-related illness among bathers and beach-goers due to pathogenic and antimicrobial-resistant enterococci (2, 36). Vancomycin resistance genes are recognized as emerging environmental contaminants (37). However, currently the standard method for identification of VRE is phenotypic characterization, disk diffusion sensitivity assay, and determination of minimum inhibitory concentration (MIC) using biochemical tests which require more time for interpretation of results (32). To overcome limitations associated with biochemical tests and conven3346

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tional PCR assays, we developed and validated an MBP based real-time PCR assay for the detection of VRE exhibiting vanA gene with the minimal hands-on time of 2.5 h. The MBprobe-based real-time PCR assay is advantageous as it does not require any post PCR analysis and melt-curve analysis thus reducing the bench-time. Further, the high annealing temperature of the MBP due to hairpin design circumvents any nonspecific probe-target hybridization and therefore reduces background fluorescence. Commercial VRE identification kits available are based on conventional biochemical tests, spectrophotometry, conventional PCR with gel electrophoresis, as well as realtime PCR assay with pre-enrichment (23, 24, 38). A major limitation of aforementioned tests is the processing time of 24-48 h. The available real-time PCR based detection kit does not specify the critical parameter of LOD necessary for evaluating the sensitivity of any real-time PCR-based strategy in pathogen detection. Other investigators have used realtime PCR assay to detect VRE isolates only in clinical samples that require 2-5 days with pre-enrichment steps (38-41). Enterococci may also adopt VBNC state, which may further limit the real-time estimation of VRE (29, 42). The conventional methods based on cultivation are biased and inefficient for determination of real pathogenic load in the environment due to poor culturability of injured and stressed organisms. None of the studies have explored the application of realtime PCR assay for detection of VBNC entities of VRE in environmental samples. Reports published in the early 1960s illustrated the occurrence, survival, and proliferation of enterococci on plants with the increase of seasonal temperature and exposure to sunlight in subtropical regions (43, 44). Similar conditions in the Indian subtropics can play a major role in greater prevalence and dissemination of pathogenic microorganisms associated with macrophytes flourishing in eutrophic river waters. The spread of E. crassipes in fresh water bodies of the Indian subcontinent has recently been reviewed (45). Further, Bhanumati et al. have shown the prevalence of antimicrobial-resistant and virulent Vibrio cholerae O139 on E. crassipes recovered from river Ganga (46). In the present study, we have demonstrated for the first time the application of molecular beacon probe based realtime quantitation for culture-independent detection of vanA gene in surface waters and E. crassipes root-mat-associated microbiota. In addition, the LOD enhancement technique helped in detection of otherwise nondetectable vanA gene concentration from the relatively clean upstream sites on river Ganga and Gomti (Tables 2 and 3). This study validated the assay with respect to VRE, vancomycin resistant variant of “enterococci”. However, it

might be possible that a very small fraction of total vanA gene concentration detected in surface water and root-matassociated microbiota was contributed by other vanA gene containing organisms. Further, the DNA extraction and purification from complex environmental matrices by modifications in boiling lysis protocol have increased the sensitivity of the quantitation method. The specificity of the assay was demonstrated by efficient amplification of purified total DNA of 4-fold diluted cultures (1 × 102 CFU/mL) of environmental enterococci isolates exhibiting VanA (n ) 25), VanB (n ) 33), and vancomycin susceptible (n ) 20) phenotype characterized in our laboratory from different surface water resources; further no amplification observed in VanB and vancomycin susceptible phenotypes (Table S2). One advantage of our MBP-based real-time PCR assay over conventional PCR is that this quantitative PCR assay in 2-fold dilution format can be used to detect very low count of cells in the sample as it takes into account statistical parameters such as correlation coefficient and PCR efficiency to set relative baseline for differentiation of spiked background against negative control. Recently, the superiority of MBP-based quantitative assay over conventional PCR for the detection of another waterborne pathogen, enterotoxigenic E. coli has been reported (28). The detection of enterococci in surface waters and aquaticplant-associated microbiota is important not only because this genus has been used as indicator of microbial quality of water but also due to their role in dissemination and persistence of antimicrobial resistance (47, 48). Earlier investigations on persistence of fecal indicator bacteria E. coli and enterococci in mats of the green alga Cladophora glomerata (L.) ku ¨ tz have concluded that such associations serve as an environmental source of indicator bacteria (30). Further multifarious use of surface waters facilitates dissemination of antimicrobial-resistant enterococci and builds a background pool of resistance (BPR) in the environment due to their transmission to plants, soil, and subsequently to the whole ecosystem by the action of transmission agents such as wild animals, insects, wind, and rain (43, 44). This BPR is sustained by plasmid maintenance system or postsegregational killing (PSK) system that secure the stable inheritance of plasmids (viz. plasmid containing vanA gene cluster) during cell division by killing or impairing the growth of cells that have lost particular plasmids (48-50). Evidently, the pervasiveness of antimicrobial-resistance in the environment leaves limited treatment options in case of fatal infections and VRE outbreaks. The prompt and early detection of the potential pathogenic and multiantimicrobial-resistant indicator bacteria such as enterococci, in surface waters can be crucial for preemptive strategy and better management of surface water resources. This study established the culture-independent, rapid, sensitive, and specific molecular beacon probe based real-time PCR strategy for detection of vanA gene in surface waters and microbiota associated with aquatic macrophyte.

Acknowledgments We are grateful to the Council of Scientific and Industrial Research (CSIR) for providing financial support for the study in the Network Project: SMM-05. The financial assistance to P.L. (SRF) and S.R. (SRF) from CSIR, Government of India, New Delhi is sincerely acknowledged. IITR manuscript no. 2721.

Supporting Information Available Methods for sample collection, total DNA isolation-purification from complex environmental samples, PCR primer and molecular beacon designing, thermal denaturation of vanA molecular beacon probe, optimization of real-time PCR assay,

list of VRE and non-VRE isolates used to validate the specificity of the assay, amplification profile and standard curves of real-time PCR experiments. This information is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) U.S. EPA. Bacterial Water Quality Standards for Recreational Waters (Freshwater and Marine Waters); EPA- 823-R-03-008; U.S. Environmental Protection Agency: Washington, DC, 2003; http://www.epa.gov/waterscience. (2) Cabelli, V.; Dufour, A. P.; McCabe, L. J.; Levin, M. A. Swimmingassociated gastroenteritis and water quality. Am. J. Epidemiol. 1982, 115, 606–616. (3) Murray, B. E.; Weinstock, G. M. Enterococci: new aspects of an old organism. Proc. Assoc. Am. Physicians 1999, 111, 328–334. (4) Bonten, M. J. M.; Willems, R.; Weinstein, R. A. Vancomycin resistant enterococci: why are they here, and where do they come from? Lancet Infect. Dis. 2001, 314–325. (5) Bell, J. M.; Paton, J. C.; Turnidge, J. Emergence of vancomycinresistant enterococci in Australia: phenotypic and genotypic characteristics of isolates. J. Clin. Microbiol. 1998, 36, 2187– 2190. (6) Mohanty, S.; Kapil, A.; Das, B. K.; Dhawan, B. Antimicrobial resistance profile of nosocomial uropathogens in a tertiary care hospital. Indian J. Med. Sci. 2003, 57, 148–154. (7) Taneja, N.; Rani, P.; Emmanuel, R.; Sharma, M. Significance of vancomycin resistant enterococci from urinary specimens at a tertiary care centre in northern India. Indian J. Med. Res. 2004, 119, 72–74. (8) Ghoshal, U.; Garg, A.; Tiwari, D. P.; Ayyagiri, A. Emerging vancomycin resistance in enterococci in India. Indian J. Pathol. Microbiol. 2006, 49, 620–622. (9) Tang, C. W.; Cheng, C. K.; Lee, T. S. Community-acquired blebrelated endophthalmitis caused by vancomycin-resistant enterococci. Can. J. Ophthalmol. 2007, 42, 477–478. (10) Moellering, R. C., Jr. Emergence of enterococcus as a significant pathogen. Clin. Infect. Dis. 1992, 14, 1173–1176. (11) Ruoff, K. L.; Maza, L. d. l.; Murtagh, M. J.; Spargo, J. D.; Ferraro, M. J. Species identities of enterococci isolated from clinical specimens. J. Clin. Microbiol. 1990, 28, 434–437. (12) Clevel, D. B. Movable genetic elements and antibiotic resistance in enterococci. Eur. J. Clin. Microbiol. Infect. Dis. 1990, 90–102. (13) Courvalin, P. Resistance of enterococci to glycopeptides. Antimicrob. Agents Chemother. 1990, 34, 2291–2296. (14) Uttley, A. H. C.; Collins, C. H.; Naidoo, J.; George, R. C. Vancomycin-resistant enterococci. Lancet 1988, i, 57–58. (15) Caplin, J. L.; Hanlon, G. W.; Taylor, H. D. Presence of vancomycin and ampicillin-resistant Enterococcus faecium of epidemic clonal complex-17 in wastewaters from the south coast of England. Environ. Microbiol. 2008, 10, 885–892. (16) Talebi, M.; Rahimi, F.; Katouli, M.; Mo¨llby, R.; Pourshafie, M. R. Epidemiological link between wastewater and human vancomycin-resistant Enterococcus faecium isolates. Curr. Microbiol. 2008, 56, 468–473. (17) Eisner, A.; Feierl, G.; Gorkiewicz, G.; Dieber, F.; Kessler, H. H.; Marth, E.; Ko¨fer, J. High prevalence of VanA-type vancomycinresistant enterococci in Austrian poultry. Appl. Environ. Microbiol. 2005, 71, 6407–6409. (18) Citak, S.; Yucel, N.; Mendi, A. Antibiotic resistance of enterococcal isolates in raw milk. J. Food Proc. Pres. 2005, 29, 183–195. (19) Ram, S.; Vajpayee, P.; Shanker, R. Prevalence of multiantimicrobial-agent resistant, shiga toxin and enterotoxin producing Escherichia coli in surface waters of river Ganga. Environ. Sci. Technol. 2007, 41, 7383–7388. (20) Cetinkaya, Y.; Falk, P.; Mayhall, C. G. Vancomycin resistant enterococci. Clin. Microbiol. Rev. 2000, 13, 686–707. (21) Arthur, M.; Courvalin, P. Genetics and mechanisms of glycopeptide resistance in enterococci. Antimicrob. Agents Chemother. 1993, 37, 1563–1571. (22) Bugg, T. D. H.; Dutka-Malen, S.; Arthur, M.; Courvalin, P.; Walsh, C. T. Identification of vancomycin resistance protein VanA as a D-alanine- D-alanine ligase of altered substrate specificity. Biochemistry 1991, 30, 2017–2021. (23) Lee, S. Y.; Park, Y. J.; Lee, G. D.; Park, J. J. Comparison of seeplex VRE detection kit with chromID VRE agar for the detection of vancomycin-resistant enterococci in faecal specimen. Clin. Microbiol. Infect. 2008, 14, S667-S759. (24) Kilic, A.; Baysallar, M.; Bahar, G.; Kucukkaraaslan, A.; Cilli, F.; Doganci, L. Evaluation of the EVIGENE VRE detection kit for detection of vanA and vanB genes in vancomycin resistant enterococci. J. Med. Microbiol. 2005, 54, 347–350. VOL. 43, NO. 9, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3347

(25) Chen, Y.; Marshall, S. A.; Winokur, P. L.; Coffman, S. L.; Wilke, W. W.; Murray, P. R.; Spiegel, C. A.; Pfaller, M. A.; Doern, G. V.; Jones, R. N. Use of molecular and reference susceptibility testing methods in a multicenter evaluation of microscan dried overnight gram-positive MIC panels for detection of vancomycin and high-level aminoglycoside resistances in enterococci. J. Clin. Microbiol. 1998, 36, 2996–3001. (26) Coombs, G. W.; Kay, I. D.; Steven, R. A.; Pearman, J. W.; Bertolatti, D.; Grubb, W. B. Should genotypic testing be done on all phenotypically vancomycin-resistant enterococci detected in hospitals? J. Clin Microbiol. 1999, 37, 1229–1230. (27) Dutka-Malen, S.; Evers, S.; Courvalin, P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin Microbiol 1995, 33, 24–27. (28) Ram, S.; Vajpayee, P.; Shanker, R. Rapid culture-independent quantitative detection of enterotoxigenic Escherichia coli in surface waters by real-time PCR with molecular beacon. Environ. Sci. Technol. 2008, 42, 4577–4582. (29) He, J. W.; Jiang, S. Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR. Appl. Environ. Microbiol. 2005, 71, 2250–2255. (30) Whitman, R. L.; Shively, D. A.; Pawlik, H.; Nevers, M. B.; Byappanahalli, M. N. Occurrence of Escherichia coli and enterococci in Cladophora (chlorophyta) in nearshore water and beach sand of Lake Michigan. Appl. Environ. Microbiol. 2003, 69, 4714–4719. (31) Matthysse, A. G.; Deora, R.; Mishra, M.; Torres, A. G. Polysaccharides cellulose, poly-β-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells. Appl. Environ. Microbiol. 2008, 74, 2384–2390. (32) Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth informational supplement M100-S15; Clinical and Laboratory Standards Institute: Wayne, PA, 2005. (33) Ke, D.; Picard, F. O. J.; Martineau, F.; Nard, C. M.; Roy, P. H.; Ouellette, M.; et al. Development of a PCR assay for rapid detection of enterococci. J. Clin. Microbiol. 1999, 37, 3497– 3503. (34) Jackson, C. R.; Fedorka-Cray, P. J.; Barrett, J. B. Use of a genus and species-specific multiplex PCR for identification of enterococci. J. Clin. Microbiol. 2004, 42, 3558–3565. (35) Khudaier, B. Y.; Tewari, R.; Shafiani, S.; Sharma, M.; Emmanuel, R.; Sharma, M.; Taneja, N. Epidemiology and molecular characterization of vancomycin resistant enterococci isolates in India. Scand. J. Infect. Dis. 2007, 39, 662–670.

3348

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 9, 2009

(36) Pruss, A. Review of epidemiological studies on health effects from exposure to recreational water. Int. J. Epidemiol. 1998, 27, 1–9. (37) Pruden, A.; Pei, R.; Storteboom, H.; Carlson, K. H. Antibiotic resistance genes as emerging contaminants: studies in northern Colorado. Environ. Sci. Technol. 2006, 40, 7445–7450. (38) Young, H. L.; Ballard, S. A.; Roffey, P. Direct detection of vanB2 using the Roche LightCycler vanA/B detection assay to indicate vancomycin-resistant enterococcal carriage-sensitive but not specific. J. Antimicrob. Chemother. 2007, 59, 809–810. (39) Palladino, S.; Kay, I. D.; Costa, A. M.; Lambert, E. J.; Flexman, J. P. Real-time PCR for the rapid detection of vanA and vanB genes. Diag. Microbiol. Infect. Dis. 2003, 45, 81–84. (40) Adams, D. N. Shortcut detection of the vanB gene cluster in enterococci by a duplex real-time PCR assay. Pathology 2006, 38, 349–352. (41) Al-Mohri, H. A.; Tadros, M. A.; Louie, L.; Vearncombe, M.; Simor, A. E. Utility of direct, real-time PCR in the management of a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (vanB genotype). Eur. J. Clin. Microbiol. Infect. Dis. 2008, 27, 321–322. (42) Lleo, M. M.; Tafi, M. C.; Canepari, P. Nonculturable Enterococcus faecalis cells are metabolically active and capable of resuming active growth. Syst. Appl. Microbiol. 1998, 21, 333–339. (43) Mundt, J. O. Occurrence of enterococci: bud, blossom, and soil studies. Appl. Microbiol. 1961, 9, 541–544. (44) Mundt, J. O. Occurrence of enterococci on plants in a wild environment. Appl. Microbiol. 1963, 11, 141–144. (45) Malik, A. Environmental challenge vis a vis opportunity: The case of water hyacinth. Environ. Int. 2007, 33, 122–138. (46) Bhanumathi, R.; Sabeena, F.; Isac, S. R.; Shukla, B. N.; Singh, D. V. Molecular characterization of Vibrio cholerae O139 Bengal isolated from water and the aquatic plant Eichhornia crassipes in the river Ganga, varanasi, India. Appl. Environ. Microbiol. 2003, 69, 2389–2394. (47) Murray, B. E. The life and times of the enterococcus. Clin. Microbiol. Rev. 1990, 3, 46–65. (48) Sletvold, H.; Johnsen, P. J.; Simonsen, G. S.; Aasnæs, B.; Sundsfjord, A.; Nielsen, K. M. Comparative DNA analysis of two vanA plasmids from Enterococcus faecium strains isolated from poultry and a poultry farmer in Norway. Antimicrob. Agents Chemother. 2007, 51, 736–739. (49) Randall, S. S.; Ward, M. P.; Maldonado, G. Can landscape ecology untangle the complexity of antibiotic resistance? Nat. Rev. Microbiol. 2006, 4, 943–952. (50) Gerdes, K.; Christensen, S. K.; Lobner-Olesen, A. Prokaryotic toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 2005, 3, 371–382.

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