Sample collection and processing
Environmental samples (water and sand) were obtained from the coasts of California and Kaua’i, Hawai’i during multiple field excursions spanning from July 2006 to March 2007 (Keymer et al. 2007; Santoro and Boehm 2007; Yamahara et al. 2007; De Sieyes et al. 2008; Knee et al. 2008). These samples (beach sands, storm drains, groundwater, estuaries, and surf zones) were chosen to assess the prevalence of the esp HF marker in a wide range of coastal environments. Mammalian and avian faecal samples from humans, dogs, horses, seagulls, seals, and sea lions were obtained from San Francisco Bay area farms, beaches, dog parks, and the Marine Mammal Center in Sausalito, California. These hosts were chosen for the study because they are common to beaches of the Pacific coast and could contribute enterococci to the coastal environment. Primary-treated sewage effluent was collected from the Palo Alto Water Quality Control Plant in Palo Alto, California.
Environmental water samples (groundwater, storm drain, estuarine, and surf zone) were screened for enterococci using either Enterolert implemented in a 97-well Quanti-tray (IDEXX, Westbrook, ME, USA) or membrane filtration onto mEI agar (Difco/BD, Sparks, MD, USA). Beach sand samples were eluted and the eluate was membrane filtered as previously described (Yamahara et al. 2007). Sewage samples were membrane-filtered onto mEnterococcus (Difco/BD) or mEI agar (EPA Method 1600). Animal faecal samples were combined with Dulbecco’s phosphate buffer saline (PBS; GIBCO/Invitrogen, Grand Island, NY, USA) to form slurries, which were then membrane-filtered in duplicate or triplicate onto mEnterococcus agar (Difco/BD) at dilution volumes producing 20–60 colonies per filter. Membrane filtration apparatuses were ultraviolet (UV)-disinfected for 5 min in a UV cross-linker (UVP, model CL1000, delivers c. 3000 μW cm−2) between samples, and filtration blanks were run.
Presumptive Enterococcus colonies from membrane filtration were enriched in trypic soy broth (TSB) as described by Scott et al. (2005). Enterococci from Enterolert Quanti-tray wells were enriched as follows: the back of the tray was sterilized with 100% ethanol, and enterococci-positive wells were aspirated using a 21½ gauge needle and syringe. Media from a single tray were combined and mixed, and then 1 ml was added to 15 ml of TSB. All TSB enrichments, regardless of the original culture media, were incubated at 41°C for 4–6 h.
Cultures of E. faecium strains E300, E470, and E734, carrying the esp gene, were graciously provided by Dr Rob Willems of University Medical Center Utrecht, The Netherlands. DNA obtained from these strains was used as template for positive controls for all esp polymerase chain reaction (PCR) assays. Staphylococcus aureus (ATCC #25923) and Streptococcus bovis (ATCC #33317) were used as negative controls from DNA extraction through PCR to ensure no cross-contamination occurred.
DNA was extracted from one millilitre of enrichment and control cultures with a QIAamp Min-Elute DNA Spin Kit (Qiagen Inc., Valencia, CA, USA), using the protocol for gram-positive bacteria. A blank experiment was performed to ensure there was no laboratory contamination at any stage of the work. Triplicate 100-μl aliquots of stationary-phase E. faecium E734 were membrane filtered, and after UV disinfection, the same filtration apparatuses were used to filter 10 ml of PBS following each aliquot of the culture. The same procedure was repeated using 1-ml aliquots of primary-treated sewage in place of the E. faecium E734. All 12 filters were placed on mENT and incubated for 48 h at 35°C. The samples were then subjected to the entire esp gene assay, from enrichment to PCR, regardless of whether there was growth on the culture media.
PCR amplification of the esp HF marker
We evaluated DNA extracts from samples described before for the presence of the esp HF marker using PCR primers described by Scott et al. (2005) (hereafter referred to as the Scott et al. primers). Each 25-μl reaction consisted of 1 × PCR buffer containing 1·5 mmol l−1 of MgCl2 (Qiagen), 0·4 μmol l−1 of each primer, 1 U of HotStar Taq (Qiagen), and 1 μl of template DNA (200–300 ng μl−1). DNase-/RNase-free water (GIBCO/Invitrogen) was used as a no-template control for each PCR run. Following an initial polymerase-activation step of 15 min at 95°C, PCR was carried out by 35 cycles of 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C, with a final extension step of 5 min at 72°C. PCR products were analysed on a 1·5% agarose gel stained with ethidium bromide, and visualized and photographed using a GelDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA) and QuantityOne software (ver. 4.6.3; Bio-Rad Laboratories).
PCR amplification of 1-kb segment of esp gene
We used a separate primer pair (forward: BL9 5′-ATTTTGCTAATGCAAGTCCA-3′; reverse: esp5R 5′-TACTGCTAAATCGGTCGTG-3′) (hereafter referred to as the 1-kb primers) to explore sequence homology in esp from various hosts and environments by sequencing a 1-kb section of the gene. The reverse primer has been used previously to sequence the esp gene (Leavis et al. 2004); we designed the forward primer such that the pair would amplify all esp genes in GenBank, from both E. faecium and E. faecalis. PCR were carried out in 25 μl reactions containing 1 × PCR buffer (Invitrogen), 2·0 mmol l−1 of MgCl2, 0·4 μmol l−1 of each primer, 0·2 mmol l−1 of dNTP, 1 U of Platinum Taq (Invitrogen), and 1·0 μl of template DNA. All control strains, as well as a no-template control were included in each PCR run. Following an initial polymerase-activation step of 4 min at 95°C, PCR was carried out by 35 cycles of 1 min at 94°C, 1 min at 54°C, and 1 min at 72°C, with a final extension step of 5 min at 72°C. PCR products were visualized on a 1·5% agarose gel stained with ethidium bromide.
esp gene clone library construction
Enterococcus faecium esp PCR products were amplified from six DNA extracts (sewage, estuarine water from Elkhorn Slough, California, and dog, horse, seal, and sea lion faeces) using the Scott et al. assay. Amplicons were gel purified using the MinElute Gel Extraction kit (Qiagen) and cloned with a TOPO-TA cloning kit, using the pCR2·1-TOPO vector and TOP10 competent cells (Invitrogen).
Eight clones from each DNA sample were randomly chosen for sequencing to confirm amplification of the desired target, and to explore the diversity of the esp gene fragment among various animal hosts and Pacific coast environments. Plasmid preps and DNA sequencing were carried out by MCLab (South San Francicso, CA, USA), using an ABI 3730XL capillary sequencer (PE Applied Biosystems, Foster City, CA).
PCR was performed using the same six DNA samples as template with the 1-kb primers. In addition, 1-kb esp PCR products were also amplified from gull faeces and surf zone seawater from Lover’s Point, California. These amplicons were cloned as described before, and 12 clones from each DNA sample were randomly chosen for sequencing using the same procedure as before.
In summary, we obtained 48 esp sequences using the Scott et al. primers, and 96 sequences using the 1-kb primers. The collection included clones from sewage, seawater, estuarine water, and dog, horse, seal, sea lion, and gull faeces (see Table 1).
Table 1. Number of esp sequences obtained from clones using two primer sets; DNA were extracted from enterococci enrichment cultures
|DNA extract||No. of clones sequenced|
|Scott et al. primers||1-kb primers||Total|
|Sea lion faeces||8||12||20|
Concentrations of enterococci in individual faecal samples were assigned the lower detection limit value (varied; on the order of 1–10 CFU g−1) if no colonies grew on the membrane filter, and the upper limit of detection (varied; range of 105–107 CFU g−1) if there were too many colonies to count. For those environmental samples that were processed using IDEXX Enterolert, an upper detection limit of 24 190 MPN per 100 ml was assigned if all wells were positive. When multiple DNA extracts from the same sample (faecal or environmental) were screened with PCR, the sample was assigned a positive result for the assay if at least one replicate produced an amplicon. Both parametric (n-way anova) and nonparametric methods (Kruskal–Wallis analysis of variance and chi-squared tests) were used to examine the variability of measured parameters between sample types. Rejection of the null hypothesis was deemed statistically significant if P < 0·05. All analyses were completed using Matlab (ver. 7.3.0 R2006b; The Mathworks, Inc., Natick, MA, USA).