• antibiotic resistance;
  • methicillin-resistant Staphylococcus aureus;
  • Panton–Valentine leukocidin;
  • SCCmec;
  • spa typing;
  • Staphylococcus aureus;
  • wastewater;
  • wastewater treatment plant


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims:  (i) To cultivate methicillin-resistant Staphylococcus aureus (MRSA) from a full-scale wastewater treatment plant (WWTP), (ii) To characterize the indigenous MRSA-flora, (iii) To investigate how the treatment process affects clonal distribution and (iv) To examine the genetic relation between MRSA from wastewater and clinical MRSA.

Methods:  Wastewater samples were collected during 2 months at four key sites in the WWTP. MRSA isolates were characterized using spa typing, antibiograms, SSCmec typing and detection of Panton–Valentine leukocidin (PVL).

Conclusions:  MRSA could be isolated on all sampling occasions, but only from inlet and activated sludge. The number of isolates and diversity of MRSA were reduced by the treatment process, but there are indications that the process was selected for strains with more extensive antibiotic resistance and PVL+ strains. The wastewater MRSA-flora had a close genetic relationship to clinical isolates, most likely reflecting carriage in the community.

Significance and Impact of the Study:  This study shows that MRSA survives in wastewater and that the WWTP may be a potential reservoir for MRSA.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to all clinically used β-lactam antibiotics (Hanssen and Ericson Sollid 2006). The only vector described for mecA, the gene encoding methicillin resistance in Staphylococcus species, is the staphylococcal cassette chromosome (SCC). MRSA has emerged as one of the most frequent causes of hospital-associated infections, and the global spread of MRSA is of major concern (Grundmann et al. 2006). Moreover, community-associated MRSA has recently emerged as an additional threat, causing infections in healthy individuals with no health care-associated risk factors (Kluytmans-Vandenbergh and Kluytmans 2006). In addition, MRSA is an increasing problem in veterinary medicine (Rich 2005), and animals can be a source of MRSA infections in humans (Lewis et al. 2008).

The role of wastewater as an environmental reservoir may be significant in development and dissemination of antibiotic resistance (Martinez 2006), particularly as a large part of prescribed antibiotics end up in the environment in an active form (Halling-Sörensen et al. 1998; Kummerer et al. 2000). This includes the β-lactam antibiotics, which have been detected in both surface waters and wastewater systems (Watkinson et al. 2009). The spread of antibiotic resistance in the environment because of horizontal gene transfer is documented (Davison 1999), and transfer of genes encoding antibiotic resistance by S. aureus in wastewater has been described in vitro (Ohlsen et al. 2003). Furthermore, mecA has been detected in both hospital and municipal wastewater (Volkmann et al. 2004; Börjesson et al. 2009a,b), and recently, we detected MRSA in municipal wastewater, using both cultivation and molecular techniques (Börjesson et al. 2009b). Thus, a potential source for dissemination and development of MRSA strains might be wastewater treatment plants (WWTPs). Further research is therefore needed to investigate the occurrence of MRSA in wastewater and the effect of the wastewater treatment processes on MRSA. In the present study, we identified MRSA in a municipal WWTP and investigated how the treatment process affected clonal distribution. In addition, we examined possible genetic relation between MRSA isolates from wastewater and clinical MRSA isolates.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The wastewater treatment plant and sampling

The municipal WWTP Ryaverket is one of the largest treatment plants in Northern Europe, receiving wastewater from nearly 830 000 person equivalents, with an average daily flow of 350 000 m3. The system has a hydraulic retention time of 8–10 h and a solid retention time of 2–4 days in the activated sludge system. It is primarily designed for biological nitrogen removal, utilizing pre-denitrification in a non-nitrifying activated sludge system, with post-nitrification in a trickling filter. A more detailed description of the treatment process can be found at

A wastewater sample of one litre was collected every second week from 20th May 2008 to 1st July 2008, at four key sites in Ryaverket WWTP: (i) inlet (IN), (ii) activated sludge (AS), (iii) after trickling filter (ATF) and (iv) outlet (OUT) (Fig. 1). The IN and OUT samples were collected from wastewater pooled over 24 h, while the samples at AS and ATF were taken as grab samples on-site in the treatment process. All samples were collected in autoclaved 1-l glass bottles (Pyrex, Sunderland, England) and stored at +4°C during sampling and transportation to the laboratory (transportation time c. 2 h). The samples were processed within 1 h after arrival.


Figure 1.  Schematic drawing of the wastewater treatment process at the municipal wastewater treatment plant Ryaverket, Gothenburg, Sweden, with the main steps indicated in boxes. Sampling sites are denoted with numbers; 1. Inlet, 2. Activated sludge, 3. After trickling filter and 4. Outlet. Modified from Börjesson et al. (2009b).

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Isolation of MRSA

One hundred and fifty millilitres of water from IN, OUT and ATF and 50 ml of water from AS were centrifuged at 8000 g for 30 min at +4°C. The pellet was resuspended in MAMSA (methicillin-aztreonam mannitol salt) broth (Nilsson et al. 2005), supplemented with 3 μg ml−1 cefoxitin and 8 μg ml−1 aztreonam, and incubated at 35°C over night. Thereafter, the broth was inoculated on Brilliance™ MRSA Agar (Oxoid, Cambridge, UK) and MRSASelect™ Chromogenic Agar (Bio-Rad Laboratories Hercules, CA, USA) and incubated at 35°C for 18–24 h. MRSA was verified using a previously described mecA and nuc Sybrgreen® real-time PCR assay (Nilsson et al. 2005), performed in an Applied Biosystems 7500 FAST Real-time PCR (Applied Biosystems, Warrington, UK). All MRSA isolates were labelled as to location (IN, AS, OUT and ATF), sample, day and month, type of MRSA agar plate (Brilliance™ = B and MRSASelect™ = S) and consecutively numbered. For example, IN1-0520B-1 was collected from inlet sample 1 on the 20th May from Brilliance™ MRSA Agar and was the first colony collected.

spa Typing, SCCmec typing and detection of PVL genes

spa Typing was performed as previously described (Harmsen et al. 2003). Sequencing of the spa gene was performed by Macrogen Inc. (Seoul, Korea), using BigDye™ chemistry, and spa types were determined using the software Ridom StaphType (Ridom GmbH, Würzburg, Germany). SCCmec typing was performed using a multiplex PCR as described by Boye et al. (2007). The PCR amplifications for spa and SCCmec typing were performed in a PTC-100TM (MJ Research Inc., Watertown, MA, USA). Detection of PVL genes was performed using a real-time PCR assay (Lina et al. 1999) in a LightCycler instrument (Roche Diagnostics, Bromma, Sweden).

Antibiotic susceptibility testing

Antibiotic susceptibility testing was performed using the disc diffusion method on ISO-sensitest agar (31·4 mg ml−1, Oxoid) according to the guidelines from the Swedish Reference Group for antibiotics ( The antibiotics tested were cefoxitin (FOX, 10 μg), erythromycin (E, 15 μg), clindamycin (DA, 15 μg), fusidic acid (FD, 10 μg), trimethoprim/sulfamethoxazole (SXT, 25 μg), tobramycin (TOB, 30 μg), vancomycin (VA, 5 μg), rifampicin (RD, 5 μg), linezolid (LZD, 10 μg), ciprofloxacin (CIP, 5 μg), trimethoprim (W, 23·75 μg + 1·25 μg) and tetracycline (TC, 30 μg). Isolates were defined as multiresistant when resistant to FOX plus at least two other classes of antibiotics.

Clonal relationship between wastewater MRSA and clinical isolates

MRSA became a mandatorily notifiable finding in Sweden in 2000, and since 2006, the Swedish Institute for Infectious Disease Control (SMI) has used spa typing as the main method for national typing of MRSA strains. The clonal relationship between spa types of MRSA isolates from wastewater and the 20 most common spa types of clinical MRSA in Sweden from 2007 to 2008 was investigated (B. Olsson-Liljequist, SMI, personal communication), using the based-upon-repeat-patterns (BURP) algorithm, with default settings in Ridom StaphType (Ridom GmbH). In 2007, 1127 cases of MRSA were reported to SMI, and in 2008 (January–November), 1217 cases were reported. The 20 most common spa types constituted 55% of all MRSA isolates in Sweden in 2007 and 60% in 2008 (B. Olsson-Liljequist, SMI, personal communication.).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

spa types and antibiograms

A total of 189 MRSA, of 29 different spa types, were isolated (Table 1, Fig. 2). According to the Ridom SpaServer (, six novel spa types (t3950, t4013, t4100, t4102, t4103 and t4140) were identified, and four additional spa types (t648, t747, t3310 and t3341) were isolated for the first time in Sweden.

Table 1. spa types and antibiograms of methicillin-resistant Staphylococcus aureus (MRSA) isolated from a municipal wastewater treatment plant
  1. R, Resistant (bold type for emphasis); I, Intermediate; S, Sensitive. Antibiotics tested: FOX, Cefoxitin; E, Erythromycin; DA, Clindamycin; FD, Fusidic acid; SXT, Trimethoprim/Sulfamethoxazol; TOB, Tobramycin; VA, Vancomycin; RD, Rifampicin; LZD, Linezolid; CIP, Ciprofloxacin; W, Trimethoprim; TC, Tetracycline.

  2. *Identical spa type with different resistance pattern.

  3. Nspa type not previously described, according to Ridom Staphtype.


Figure 2.  The number of methicillin-resistant Staphylococcus aureus isolates and their spa types in the wastewater treatment process at the wastewater treatment plant. t127/t127* denotes identical spa type with different antibiograms. I, inlet; A, activated sludge. bsl00001, 5/20-2008; bsl00036, 6/3-2008; bsl00077, 6/17-2008; □, 7/1-2008.

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Antibiotic resistance patterns for each spa type are listed in Table 1. All isolates of the same spa type had identical antibiograms, except for isolates of the spa type t127, which showed two different antibiograms. Resistance to β-lactams exclusively was noted in 12 spa types, which accounted for 65% of total isolates. Nine spa types were resistant to at least two different classes of antibiotics, in addition to FOX, and hence were classified as multiresistant. One of the novel spa types, t4140, was multiresistant.

SCCmec typing and detection of PVL genes

From each sampling site and occasion, one prototype isolate regarding spa type and antibiogram was selected for SCCmec typing and PVL detection (Table 2). Only SCCmec types I (31%) and IV (69%) were identified, with identical prototypes carrying the same SCCmec (Table 2). Of the twelve spa types exclusively resistant to FOX, eight carried SCCmec IV. The spa type t127 resistant to E/DA and TE carried SCCmec I, while t127 resistant to E/DA and FD carried SCCmec IV.

Table 2.   SCCmec typing and detection of PVL in prototype isolates (sharing identical spa type and antibiogram) cultivated from wastewater
Sampling dateIsolatespa TypeSCCmecPVL
  1. IN, inlet water; AS, activated sludge, PVL, Panton–Valentine leukocidin.

  2. *Identical spa type with different resistance pattern.


The spa types t008 (SCCmec I), t019 (SCCmec IV), t127 (SCCmec I) and t852 (SCCmec IV) carried PVL genes (PVL+). But the t127 (SCCmec IV) was PVL–.

Clonal relationship of wastewater MRSA and clinical MRSA

Eight spa types isolated from the wastewater: t002, t008, t015, t018, t019, t127, t186 and t790 were represented among the 20 most common, clinically isolated MRSA spa types in Sweden (2007–2008) (Table 3). In addition, the spa types t067 and t172 were among the 30 most reported in the Ridom SpaServer ( The remaining 19 spa types either belonged to the same spa clonal complexes (spa-CCs) as the 20 most common spa types in Sweden (2007–2008) (Table 3, Fig. 3) or had been described as MRSA previously in Sweden according to the Ridom SpaServer. Five of six novel spa types belonged to spa-CCs, including clinical MRSA, of which t4013 and t4103 belonged to the same spa-CC (CC186).

Table 3.   Cluster analysis of spa types, using based-upon-repeat-patterns (BURP), of methicillin-resistant Staphylococcus aureus (MRSA) from municipal wastewater and the 20 most common clinical isolates in Sweden 2007–2008
Clusterspa TypeRepeat succession*Origin
  1. CC, Clonal Complex; IN, Inlet; AS, Activated sludge.

  2. *Repeat succession by Ridom nomenclature

  3. †C--/-- = Clinical isolates year/number of isolates (B. Olsson-Liljequist, personnel communication)

  4. ‡No founder identified.

  5. §Excluded from analysis by BURP because spa type shorter than five repeats.

  6. Nspa type described for the first time in this study according to Ridom Staphtype.

t00811-19-12-21-17-34-24-34-22-25AS, C07/118†, C08/103
t02411-12-21-17-34-24-34-22-25C07/19, C08/29
spa-CC012t01908-16-02-16-02-25-17-24AS, C07/16, C08/45
t01815-12-16-02-16-02-25-17-24-24-24IN, AS, C07/11
t03715-12-16-02-25-17-24C07/31, C08/32
spa-CC437/441t43704-20-17-20-17-25-34C07/23, C08/21
t17204-20-17-45-16-34IN, AS
t18607-12-21-17-13-13-34-34-33-34IN, AS, C08/13
t69007-12-21-17-13-13-34-34-34-33-34C07/24, C08/18
ClusterY‡t00326-17-20-17-12-17-17-16C07/10, C08/13
t00226-23-17-34-17-20-17-12-17-16IN, C07/75, C08/109
t03226-23-23-13-23-31-29-17-31-29-17-25-17-25-16-28C07/102, C08/44
t00526-23-13-23-31-05-17-25-17-25-16-28C07/10, C08/12
t79026-23-13-23-31-29-17-25-17-25-16-28IN, C08/11
Singletont01508-16-02-16-34-13-17-34-16-34IN, C07/27, C08/26
Singletont04407-23-12-34-34-33-34C07/102, C08/93
Singletont12707-23-21-16-34-33-13IN, AS, C07/21, C08/30

Figure 3.  Dendogram based on cluster analysis of methicillin-resistant Staphylococcus aureus (MRSA) spa types isolated from the municipal wastewater treatment plant and the 20 most common MRSA spa types in Sweden 2007–2008, using based-upon-repeat-patterns (BURP) with default settings in Ridom StaphType. spa type number within frames denotes spa types isolated from municipal wastewater.

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MRSA during the wastewater treatment process

MRSA was isolated exclusively from IN and AS, with 66% of the isolates cultivated from IN (Fig. 2). There were 19 and 15 spa types isolated from IN and AS, respectively. The majority of spa types, 76%, were only detected in one sampling occasion and in one sampling site (Fig. 2). The spa type t172 was most frequently identified, accounting for 29% of the isolates and occurred on three and two sampling occasions in IN and AS, respectively. The spa types t018, t127, t186, t386 and t3341 were isolated on more than one sampling occasion (Fig. 2). Four of the six novel spa types: t4013, t4102, t4103 and t4140 were isolated exclusively from AS. In the AS, 40% of the spa types were defined as multiresistant, while in IN only 21% were multiresistant. All PVL+ isolates were detected in AS (Table 2).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

In the current study, we detected a high number of MRSA of genetically diverse spa types in municipal wastewater (Fig. 3, Table 3), showing that it was not a specific lineage that survived in this environment. This extended the results of our recent study (Börjesson et al. 2009b), wherein MRSA, of three spa types, was cultivated from untreated municipal wastewater for the first time. Our findings contrast with previous studies, which have shown that S. aureus either has a low prevalence or does not occur in wastewater (Schwartz et al. 2003; Volkmann et al. 2004; Savichtcheva et al. 2007; Shannon et al. 2007). A possible explanation for our detection of MRSA could be the use of our optimized cultivation protocol (Börjesson et al. 2009b). We had expected a low prevalence of MRSA in wastewater, given that Sweden has a low clinical prevalence of MRSA (SWEDRES 2007). The results may, therefore, indicate that WWTPs are a potential reservoir for MRSA. This is further supported by the finding of six novel spa types in the wastewater, of which four were isolated only in AS, possibly indicating the evolution of new strains in the wastewater. However, it is also likely that previously uncharacterized MRSA strains are circulating in the community. At any rate, it is likely that a large part of the MRSA isolated from this wastewater originates from the population connected to the WWTP, as most of the spa types have been isolated previously in clinical settings in Sweden (Table 3). Furthermore, the wastewater MRSA flora changed with each sampling occasion, indicating that the findings are partly dependent upon the recent flora transiting the WWTP. In addition, the wastewater MRSA had a high genetic diversity, harboured mostly SCCmec IV and was resistant only to β-lactam antibiotics, which is comparable to clinical isolates found in Scandinavia (Berglund et al. 2005, 2009; Bartels et al. 2007). However, there are indications that some MRSA strains may be resident in the WWTP and/or better adapted to the wastewater environment, e.g. isolates of spa type t172. The t172 isolates were found on multiple sampling occasions, both in IN and in AS, and was the most retrieved spa type (Fig. 2). It was also found in the same WWTP during the previous study in November 2007 (Börjesson et al. 2009b).

The wastewater treatment process appears to have a negative impact on survival of MRSA, as more isolates and spa types could be isolated from IN when compared to AS, and no MRSA could be isolated from OUT and ATF. However, the absence of MRSA in OUT or ATF may also be explained by S. aureus entering an unculturable, but viable state, in wastewater (Ohlsen et al. 2003). However, the current results are in line with those from our previous study, in which MRSA was detected using real-time PCR primarily in the early treatment steps (Börjesson et al. 2009b). Although the WWTP reduced the diversity of MRSA, there are indications that a selection of strains with more extensive antibiotic resistance occurs, as the ratio of multiresistant MRSA in the AS was higher compared to the ratio in IN. A selection for PVL+ strains was also noted, as they were only detected in the AS. PVL is a marker of increased virulence (Boyle-Vavra and Daum 2007), and a possible effect of the wastewater treatment process on PVL and other virulence markers is of major concern. That the wastewater MRSA isolates harboured only SCCmec I and IV also suggests MRSA selection in and during transportation to the WWTP. The frequent occurrence of SCCmec IV could be expected, because it dominates in MRSA isolated in Scandinavia (Berglund et al. 2005; Hanssen et al. 2005; Larsen et al. 2009) and because carriage of SCCmec IV does not impose an energetic cost (Lee et al. 2007). The fact that SCCmec II and III could not be detected may be because they are energetically unfavourable and because of their carriage of additional antibiotic resistance genes and larger size (Deurenberg and Stobberingh 2008). This may also explain the selection of SCCmec I, which is smaller in size than types II and III and because it does not carry any additional resistance genes (Deurenberg and Stobberingh 2008)). However, the absence of SCCmec V, which is similar in size to IV and does not carry any additional antibiotic resistance genes (Deurenberg and Stobberingh 2008), is hard to explain with this model.

Our study shows that MRSA strains can be found over time in municipal wastewater. We also show that wastewater treatment processes reduce the diversity as well as the number of MRSA strains, but simultaneously select for strains with more extensive antibiotic resistance and PVL+ strains. In addition, a number of novel spa types are detected in the wastewater. These results suggest that MRSA in wastewater environments can represent an unrecognized health threat.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

We thank Ann Mattsson, Gryab, Gothenburg, Sweden, for support and fruitful discussions and Lucica Enache and Åsa Nilsson, Gryab, for sampling at Ryaverket. Michael Toepfer is acknowledged for comments and linguistic revision of the manuscript. We are grateful to Kit Boye, Hvidovre Hospital, Hvidovre, Denmark, for providing DNA from S. aureus with known SCCmec types. Financial support was provided by the Swedish Research Council for Environment, Agriculture Science and Spatial Planning (Formas, contract no 245-2005-860), the Medical Research Council of South Eastern Sweden (FORSS) and the County Council of Östergötland, Sweden.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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