Study sites. Four WWTPs were included in this study: two in the Mid-Atlantic region and two in the Midwest. The treatment steps and sampling locations at each of the treatment plants are illustrated in Figure 1 .
Mid-Atlantic WWTP1 (Figure 1A ) is a tertiary WWTP in an urban area that processes 681,390 m3/day of wastewater, with a peak capacity of 1.51 million m3/day. Mid-Atlantic WWTP2 (Figure 1B ), a tertiary WWTP in a suburban area, processes 7,570 m3/day of wastewater and has a peak capacity of 45,425 m3/day. Tertiary wastewater treatment includes primary treatment (physical removal of solids), secondary treatment (biological treatment), and additional treatment that can include, but is not limited to, chlorination, ultraviolet radiation, or filtration. The incoming wastewater (influent) at both Mid-Atlantic plants includes domestic and hospital wastewater, and effluent (discharge) from both Mid-Atlantic plants is piped to landscaping sites for reuse in spray irrigation.
Midwest WWTP1 (Figure 1C ) is a tertiary WWTP in a rural area that processes 1,363 m3/day of wastewater, with a peak capacity of 10,978 m3/day. The incoming water includes domestic wastewater and agriculturally influenced stormwater. Seasonal chlorination occurs in June, July, and August, and chlorinated effluent is piped to a landscaping site for reuse in spray irrigation. Midwest WWTP2 (Figure 1D ), a secondary WWTP (with no on-site disinfection) in a rural area, processes 1,439 m3/day and has a peak capacity of 7,571 m3/day. Secondary wastewater treatment includes only primary treatment (physical removal of solids) and secondary treatment (biological treatment). The incoming water at this plant includes domestic wastewater, wastewater from a food production facility, and agriculturally influenced stormwater. Unchlorinated effluent is piped to an agricultural site for crop irrigation.
Sample collection. A total of 44 grab samples were collected between October 2009 and October 2010: 12 samples from Mid-Atlantic WWTP1; 8 from Mid-Atlantic WWTP2; 12 from Midwest WWTP1; and 12 from Midwest WWTP2. The timing of each sampling event was determined by the availability and schedule of the WWTP operators. The sampling time schedule and specific sampling locations for each plant are indicated inTables 1 and 2 and Figure 1 . Samples were collected in 1-L sterile polyethylene Nalgene® Wide Mouth Environmental Sample Bottles (Nalgene, Lima, OH), labeled, and transported to the laboratory at 4°C. All samples were processed within 24 hr.
Isolation. Membrane filtration was used to recover S. aureus and MRSA from wastewater samples. Briefly, 300 mL of each sample were vacuum filtered through a 0.45-µm, 47-mm mixed cellulose ester filter (Millipore, Billerica, MA). Filters were then enriched in 40 mL of m Staphylococcus broth (Becton, Dickinson and Company, Franklin Lakes, NJ), vortexed, and incubated at 37°C for 24 hr. A 10-µL loopful of each enrichment was then plated in duplicate on MRSASelect (Bio-Rad Laboratories, Hercules, CA) and Baird Parker agar (Becton, Dickinson and Company) for the isolation of MRSA and total S. aureus, respectively. Plates were incubated at 37°C for 24 hr. Resulting black colonies with halos on Baird Parker agar and hot pink colonies on MRSASelect were considered presumptive S. aureus and MRSA, respectively. These colonies were purified on Brain Heart Infusion (BHI) agar (Becton, Dickinson and Company) and archived in Brucella broth (Becton, Dickinson and Company) with 15% glycerol at –80°C. For quality control and quality assurance throughout the isolation process, S. aureus ATCC 43300 [American Type Culture Collection (ATCC), Manassas, VA] was used as a positive control and phosphate-buffered saline was used as a negative control.
Identification. S. aureus and MRSA were confirmed using Gram stain, the coagulase test (Becton, Dickinson and Company), the catalase test, and polymerase chain reaction (PCR). DNA extraction was carried out using the MoBio UltraClean® Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) following the manufacturer’s recommendations. For confirmation of S. aureus, we carried out PCR amplification of the S. aureus-specific nuc gene using NUC1 and NUC2 primers (Fang and Hedin 2003 (link)). For MRSA differentiation, we performed PCR amplification targeting the mecA gene, which encodes for methicillin resistance, using ECA1 and MECA2 primers, as previously described by Fang and Hedin (Brakstad et al. 1992 (link); Fang and Hedin 2003 (link); Smyth et al. 2001 (link)). The method was modified by including an internal control, using primers targeting the 16S rDNA genes, in a multiplex PCR assay (Edwards et al. 1989 (link)). PCR amplification consisted of an initial denaturing step of 95°C for 3 min, followed by 34 cycles of denaturing at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec, with a final extension at 72°C for 5 min.
Antimicrobial susceptibility testing. We performed antimicrobial susceptibility testing on all PCR-confirmed MRSA (n = 240) and MSSA (n = 119) isolates using the Sensititre® microbroth dilution system (Trek Diagnostic Systems Inc., Cleveland, OH) in accordance with the manufacturer’s instructions. Overnight cultures were transferred to sterile demineralized water (Trek Diagnostic Systems) to achieve a 0.5 McFarland standard. Then, 30 µL of each suspension was transferred to sterile cation-adjusted Mueller Hinton broth (Trek Diagnostic Systems) and 50 µL of the broth solution was then dispensed into GPN3F minimal inhibitory concentration (MIC) plates (Trek Diagnostic Systems Inc.) with the following antibiotics: erythromycin (ERY; 0.25–4 µg/mL), clindamycin (CLI; 0.12–2 µg/mL), quinupristin/dalfopristin (SYN; 0.12–4 µg/mL), daptomycin (DAP; 0.25–8 µg/mL), vancomycin (VAN; 1–128 µg/mL), tetracycline (TET; 2–16 µg/mL), ampicillin (AMP; 0.12–16 µg/mL), gentamicin (GEN; 2–16, 500 µg/mL), levofloxacin (LEVO; 0.25–8 µg/mL), linezolid (LZD; 0.5–8 µg/mL), ceftriaxone (AXO; 8–64 µg/mL), streptomycin (STR; 1,000 µg/mL), penicillin (PEN; 0.06–8 µg/mL), rifampin (RIF; 0.5–4 µg/mL), gatifloxacin (GAT; 1–8 µg/mL), ciprofloxacin (CIP; 0.5–2 µg/mL), trimethoprim/sulfamethoxazole (SXT; 1/19–4/76 µg/mL), and oxacillin+2%NaCl (OXA+; 0.25–8 µg/mL). Enterococcus faecalis ATCC 29212 and S. aureus ATCC 29213 strains were used for quality control. MICs were recorded as the lowest concentration of an antimicrobial that completely inhibited bacterial growth [Clinical and Laboratory Standards Institute (CLSI) 2010]. Resistance break points published by the CLSI were used (CLSI 2010). Multidrug resistance (MDR) was defined as resistance to two or more classes of antibiotics.
SCCmec typing. We used a multiplex PCR assay developed by Milheiriço et al. (2007) (link) to characterize the MRSA isolates (n = 240) by SCCmec type (Milheiriço et al. 2007 (link); Oliveira and de Lencastre 2002 (link)). SCCmec strains COL (type I), BK2464 (type II), ANS46 (type III), MW2 (type IVa), HAR22 (type IVh), and HDE288 (type VI) were used as positive controls for SCCmec typing.
PVL screening. All MRSA isolates, confirmed by possession of the nuc and mecA genes by PCR and an identifiable SCCmec type (n = 236), were screened for PVL by PCR of the pvl gene according to Strommenger et al. (2008) (link). S. aureus ATCC strain 25923 was used as a positive control.
PFGE. We performed PFGE on a subset of 22 MRSA isolates. To ensure a diverse, representative subset, isolates were selected using the following criteria: treatment plant, sampling date, SCCmec type, and each sampling location that had a positive sample. PFGE was based on the Centers for Disease Control and Prevention (CDC) Laboratory Protocol for Molecular Typing of S. aureus by PFGE (CDC 2011 ). We used SmaI (Promega, Madison, WI) to digest genomic DNA. Digested samples were run in 1% SeaKem® Gold agarose gels (Cambrex Bio Science Rockland Inc., Rockland, ME) in 0.5X TBE (tris-borate- EDTA) using a CHEF Mapper (Bio-Rad) for 18.5–19 hr at 200 V, 14°C, and initial and final switch of 5 and 40 sec. Cluster analysis was performed using BioNumerics software v5.10 (Applied Maths Scientific Software Development, Saint-Martens-Latem, Belgium) using Dice coefficient and the unweighted pair-group method. Optimization settings for dendrograms were 1.0% with a position tolerance of 0.95%. Based on the similarity of the control strains, isolates were considered clones if similarity was ≥ 88%. Salmonella serotype Braenderup strain H9812 was used as the standard. PFGE strain types were compared with USA types (100, 200, 300, 400, 500, 600, 700, 800, 1000, and 1100).
Statistical analyses. Descriptive statistics include the percentages of wastewater samples positive for MRSA (Table 1 ) and MSSA (Table 2 ) by WWTP. Because PFGE was not performed on all isolates, statistical analyses of antibiotic resistance data were limited to MRSA (n = 84) and MSSA (n = 58) isolates expressing unique phenotypic profiles; this allowed us to reduce bias that could be introduced by including clones. Two-sample tests of proportions were performed between MRSA and MSSA isolates with respect to the percent resistance of each group of isolates to each of the 18 tested antibiotics. Analysis of variance was then used to compare the average numbers of antibiotics against which MRSA and MSSA isolates were resistant. In all cases, p-values ≤ 0.05 were defined as statistically significant. All statistical analyses were performed using Stata/IC 10 (StataCorp LP, College Station, TX) and SAS 9.2 (SAS Institute Inc., Cary, NC).
Mid-Atlantic WWTP1 (
Midwest WWTP1 (
Sample collection. A total of 44 grab samples were collected between October 2009 and October 2010: 12 samples from Mid-Atlantic WWTP1; 8 from Mid-Atlantic WWTP2; 12 from Midwest WWTP1; and 12 from Midwest WWTP2. The timing of each sampling event was determined by the availability and schedule of the WWTP operators. The sampling time schedule and specific sampling locations for each plant are indicated in
Isolation. Membrane filtration was used to recover S. aureus and MRSA from wastewater samples. Briefly, 300 mL of each sample were vacuum filtered through a 0.45-µm, 47-mm mixed cellulose ester filter (Millipore, Billerica, MA). Filters were then enriched in 40 mL of m Staphylococcus broth (Becton, Dickinson and Company, Franklin Lakes, NJ), vortexed, and incubated at 37°C for 24 hr. A 10-µL loopful of each enrichment was then plated in duplicate on MRSASelect (Bio-Rad Laboratories, Hercules, CA) and Baird Parker agar (Becton, Dickinson and Company) for the isolation of MRSA and total S. aureus, respectively. Plates were incubated at 37°C for 24 hr. Resulting black colonies with halos on Baird Parker agar and hot pink colonies on MRSASelect were considered presumptive S. aureus and MRSA, respectively. These colonies were purified on Brain Heart Infusion (BHI) agar (Becton, Dickinson and Company) and archived in Brucella broth (Becton, Dickinson and Company) with 15% glycerol at –80°C. For quality control and quality assurance throughout the isolation process, S. aureus ATCC 43300 [American Type Culture Collection (ATCC), Manassas, VA] was used as a positive control and phosphate-buffered saline was used as a negative control.
Identification. S. aureus and MRSA were confirmed using Gram stain, the coagulase test (Becton, Dickinson and Company), the catalase test, and polymerase chain reaction (PCR). DNA extraction was carried out using the MoBio UltraClean® Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) following the manufacturer’s recommendations. For confirmation of S. aureus, we carried out PCR amplification of the S. aureus-specific nuc gene using NUC1 and NUC2 primers (Fang and Hedin 2003 (link)). For MRSA differentiation, we performed PCR amplification targeting the mecA gene, which encodes for methicillin resistance, using ECA1 and MECA2 primers, as previously described by Fang and Hedin (Brakstad et al. 1992 (link); Fang and Hedin 2003 (link); Smyth et al. 2001 (link)). The method was modified by including an internal control, using primers targeting the 16S rDNA genes, in a multiplex PCR assay (Edwards et al. 1989 (link)). PCR amplification consisted of an initial denaturing step of 95°C for 3 min, followed by 34 cycles of denaturing at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec, with a final extension at 72°C for 5 min.
Antimicrobial susceptibility testing. We performed antimicrobial susceptibility testing on all PCR-confirmed MRSA (n = 240) and MSSA (n = 119) isolates using the Sensititre® microbroth dilution system (Trek Diagnostic Systems Inc., Cleveland, OH) in accordance with the manufacturer’s instructions. Overnight cultures were transferred to sterile demineralized water (Trek Diagnostic Systems) to achieve a 0.5 McFarland standard. Then, 30 µL of each suspension was transferred to sterile cation-adjusted Mueller Hinton broth (Trek Diagnostic Systems) and 50 µL of the broth solution was then dispensed into GPN3F minimal inhibitory concentration (MIC) plates (Trek Diagnostic Systems Inc.) with the following antibiotics: erythromycin (ERY; 0.25–4 µg/mL), clindamycin (CLI; 0.12–2 µg/mL), quinupristin/dalfopristin (SYN; 0.12–4 µg/mL), daptomycin (DAP; 0.25–8 µg/mL), vancomycin (VAN; 1–128 µg/mL), tetracycline (TET; 2–16 µg/mL), ampicillin (AMP; 0.12–16 µg/mL), gentamicin (GEN; 2–16, 500 µg/mL), levofloxacin (LEVO; 0.25–8 µg/mL), linezolid (LZD; 0.5–8 µg/mL), ceftriaxone (AXO; 8–64 µg/mL), streptomycin (STR; 1,000 µg/mL), penicillin (PEN; 0.06–8 µg/mL), rifampin (RIF; 0.5–4 µg/mL), gatifloxacin (GAT; 1–8 µg/mL), ciprofloxacin (CIP; 0.5–2 µg/mL), trimethoprim/sulfamethoxazole (SXT; 1/19–4/76 µg/mL), and oxacillin+2%NaCl (OXA+; 0.25–8 µg/mL). Enterococcus faecalis ATCC 29212 and S. aureus ATCC 29213 strains were used for quality control. MICs were recorded as the lowest concentration of an antimicrobial that completely inhibited bacterial growth [Clinical and Laboratory Standards Institute (CLSI) 2010]. Resistance break points published by the CLSI were used (CLSI 2010). Multidrug resistance (MDR) was defined as resistance to two or more classes of antibiotics.
SCCmec typing. We used a multiplex PCR assay developed by Milheiriço et al. (2007) (link) to characterize the MRSA isolates (n = 240) by SCCmec type (Milheiriço et al. 2007 (link); Oliveira and de Lencastre 2002 (link)). SCCmec strains COL (type I), BK2464 (type II), ANS46 (type III), MW2 (type IVa), HAR22 (type IVh), and HDE288 (type VI) were used as positive controls for SCCmec typing.
PVL screening. All MRSA isolates, confirmed by possession of the nuc and mecA genes by PCR and an identifiable SCCmec type (n = 236), were screened for PVL by PCR of the pvl gene according to Strommenger et al. (2008) (link). S. aureus ATCC strain 25923 was used as a positive control.
PFGE. We performed PFGE on a subset of 22 MRSA isolates. To ensure a diverse, representative subset, isolates were selected using the following criteria: treatment plant, sampling date, SCCmec type, and each sampling location that had a positive sample. PFGE was based on the Centers for Disease Control and Prevention (CDC) Laboratory Protocol for Molecular Typing of S. aureus by PFGE (CDC 2011 ). We used SmaI (Promega, Madison, WI) to digest genomic DNA. Digested samples were run in 1% SeaKem® Gold agarose gels (Cambrex Bio Science Rockland Inc., Rockland, ME) in 0.5X TBE (tris-borate- EDTA) using a CHEF Mapper (Bio-Rad) for 18.5–19 hr at 200 V, 14°C, and initial and final switch of 5 and 40 sec. Cluster analysis was performed using BioNumerics software v5.10 (Applied Maths Scientific Software Development, Saint-Martens-Latem, Belgium) using Dice coefficient and the unweighted pair-group method. Optimization settings for dendrograms were 1.0% with a position tolerance of 0.95%. Based on the similarity of the control strains, isolates were considered clones if similarity was ≥ 88%. Salmonella serotype Braenderup strain H9812 was used as the standard. PFGE strain types were compared with USA types (100, 200, 300, 400, 500, 600, 700, 800, 1000, and 1100).
Statistical analyses. Descriptive statistics include the percentages of wastewater samples positive for MRSA (
Full text: Click here