Abstract

Multidrug-resistant Staphylococcus aureus strains have been commonly found in hospitals and communities causing wide ranges of infections among humans and animals. Typing of these strains is a key factor to reveal their clonal dissemination in different regions. We investigated the prevalence and dissemination of different clonal groups of S. aureus with resistance phenotype to multiple antibiotics in two sewage treatment plants (STPs) in Tehran, Iran over four sampling occasions. A total of 576 S. aureus were isolated from the inlet, sludge and outlet. Of these, 80 were identified as methicillin-resistant S. aureus (MRSA) and were further characterized using a combination of Phene Plate (PhP) typing, staphylococcal cassette chromosome mec (SCCmec), ccr types, prophage and antibiotic-resistant profiling. In all, eight common type (CT) and 13 single PhP type were identified in both STPs, with one major CT accounting for 38.8% of the MRSA strains. These strains belonged to three prophage patterns and five prophage types with SCCmec type III being the predominant type. Resistance to 11 out of the 17 antibiotics tested was significantly (P < 0.0059) higher among the MRSA isolates than methicillin-sensitive S. aureus (MSSA) strains. The persistence of the strains in samples collected from the outlet of both STPs was 31.9% for MRSA and 23.1% for MSSA. These data indicated that while the sewage treatment process, in general, is still useful for removing most MRSA populations, some strains with SCCmec type III may have a better ability to survive the STP process.

HIGHLIGHTS

  • Survival of MRSA in STPs has a major impact on public health if they enter surface waters.

  • Here we show that certain clones of MRSA carrying SCCmec type III have a better ability to survive treatment stages of the STPs.

  • The fact that these clones were resistant to up to 12 antibiotics suggests that these clones may also have a better ability to either gain or retain antibiotic-resistant genes during the STP process.

INTRODUCTION

Staphylococcus aureus is responsible for a wide range of human infections in hospitals and communities (Mounier et al. 2017). These strains are often resistant to several antibiotics, includ­ing methicillin (Hanssen & Ericson Sollid 2006). The first methicillin-resistant S. aureus (MRSA) was discovered in 1960, and since then the number of diseases caused by both hospital- (HA) and community-acquired (CA) MRSA has increased (Goldstein et al. 2012). In 1999, the emergence of CA-MRSA in groups of people with close physical contact, as an important risk factor, was reported, indicating that the epidemiology of MRSA is changing (Charlebois et al. 2004; Bassetti et al. 2009). To date, 13 different types of staphylococcal cassette chromosome mec (SCCmec) have been reported among HA-MRSA, CA-MRSA and livestock-associated MRSA strains, in which CA-MRSA strains often harbor SCCmec types IV, V or VII, encoding a pore-forming cytotoxin Panton–Valentine leucocidin (PVL), and are susceptible to most non-β-lactam antimicrobial agents (Otter & French 2011, 2012). Typing of MRSA strains based on the presence of different SCCmec types is a useful epidemiological tool and a key element to reveal their clonal dissemination in different regions. On the other hand, different typing methods such as pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, prophage typing and Phene Plate (PhP) biochemical fingerprinting also have been used extensively in the epidemiology of MRSA strains (Javidnia et al. 2013; Rahimi et al. 2014; Goudarzi et al. 2016; Hashemizadeh et al. 2019).

The control of CA-MRSA infections is very important and requires the identification of environmental reservoirs of these strains. Wastewater could be a possible source of MRSA strains in communities and may have a significant role in the development and dissemination of antibiotic resistance (Börjesson et al. 2009b). Methicillin resistance genes have been detected in sewage treatment plants (STPs) in Sweden (Börjesson et al. 2009a). Allen et al. (2010) showed the role of horizontal gene transfer in the spread of antibiotic resistance in the environment. Börjesson et al. (2010) and Goldstein et al. (2012) identified mecA as the gene responsible for methicillin resistance in MRSA isolates, initially in STPs. These findings indicate that the wastewater could be the source and cause of the spread of MRSA in communities (Iwane et al. 2001).

In the present study, we investigated the prevalence of different clonal groups of multiple drug-resistant MRSA and methicillin-sensitive S. aureus (MSSA) strains in two urban STPs in Tehran, Iran. We also assessed the persistence of MRSA strains by comparing strains isolated from the inlet, sludge and the outlet of the STPs.

METHODS

Sites and sample collection

Between July 2015 and June 2016, a total of 24 samples were collected over four sampling occasions from two STPs (12 samples from each STP) located in the north and west of Tehran, Iran and receive sewage and wastewater from households, commercial areas and hospitals within the north and west parts of the Tehran metropolitan area. On each occasion, samples were collected from the inlet (incoming raw sewage), sludge and outlet (outgoing-treated waste) of each STP after chlorination. Treatment stages of municipal wastes at both STPs were similar and included a primary treatment by physical removal of solids, secondary treatment by biological treatment and additional treatment of wastewater by chlorination. Samples were collected in 500 ml sterile bottles using the ‘grab-sampling’ technique. All samples were transported to the laboratory on ice and processed within 3 h of collection.

Isolation and identification of S. aureus

Serial dilutions were prepared from each sample, and 500 ml of each diluted sample was filtered through a 0.45-μm filter membrane (Millipore Corporation, Burlington, MA, USA). Filters were placed on Baird Parker (BP) agar (Merck, Darmstadt, Germany) without antibiotics, plates were incubated for 24 h at 37 °C and black colonies with halos were confirmed as S. aureus using species-specific primers for nucA gene by polymerase chain reaction (PCR) (Rahimi et al. 2014).

Antibiotic susceptibility tests

The susceptibility of all S. aureus isolates to 18 antibiotics was assessed using the disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standard Institute 2016). All antibiotics disks were purchased from Rosco (Taastrup, Denmark) and included cefoxitin (30 μg), amikacin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), erythromycin (15 μg), gentamicin (10 μg), kanamycin (30 μg), linezolid (30 μg), minocycline (30 μg), nitrofurantoin (50 μg), penicillin (10 U), quinupristin-dalfopristin (15 μg), rifampin (5 μg), tobramycin (10 μg), trimethoprim-sulfamethoxazole (1.25–23.75 μg) and tetracycline (30 μg). E-test strips (AB, Biomerieux, France) were used to identify the minimum inhibitory concentrations (MICs) of vancomycin-resistant strains. Cefoxitin-resistant strains were confirmed as MRSA using the mecA gene primer as described previously (Rahimi et al. 2014), and their MICs were determined using oxacillin E-test strips.

PhP typing

All S. aureus isolates were initially typed using a high-resolution biochemical fingerprinting method, the PhP-CS plate (PhPlate AB, Stockholm, Sweden), which has been specifically developed for typing of staphylococci. Each plate is comprised of four sets of 24 highly discriminatory substrates to differentiate staphylococcal strains (Thompson et al. 2013). In brief, heavy inoculum consisting of a loopful of a fresh bacterial culture was prepared in 8 ml of growth media containing 0.05% (w/v) yeast extract, 0.5% (w/v) NaCl, 0.2% (w/v) proteose peptone and 0.011% (w/v) bromothymol blue. Using a multichannel pipette, all 24 wells of each set were inoculated with 150 μl of each bacterial suspension. The plates were then incubated at 37 °C for 64 h, and images of the plates were scanned after 16, 40 and 64 h (Thompson et al. 2013) with a HP Scanjet 4,890 scanner (Hewlett-Packard, Palo Alto, CA, USA). Using the PhPlate software (PhPWin ver. 4.2; PhP Microplate Techniques AB), all scanned images were converted to numerical values, and the mean of three readings was calculated to produce a biochemical fingerprint for each isolate. Similarity among the isolates was calculated as correlation (similarity) coefficient after a pairwise comparison of the biochemical fingerprints and clustered according to the unweighted pair group method with arithmetic averages to yield a dendrogram (Sneath & Sokal 1973). Isolates having identical fingerprints were regarded as belonging to the same PhP type and assigned to a common type (CT).

DNA extraction and confirmation of MRSA

The boiling method was used for DNA extraction from S. aureus isolates. Briefly, one isolated colony from each plate was transferred to 200 μl distilled water and boiled for 15 min at 100 °C. The mixture was centrifuged at 13,000 × g for 15 min, and 10 μl of supernatant was used as the DNA template in the PCR mixture. DNA extraction from cefoxitin-resistant isolates was carried out using the High Pure PCR Template Preparation Kit (Roche, Germany) according to the manufacturer's instructions. Nanodrop 1000 (NanoDrop, Wilmington, NC, USA) was used to evaluate the concentration of all extracted DNA.

SCCmec and ccr typing

SCCmec typing and ccr typing were performed as described previously by Zhang et al. (2005). Briefly, SCCmec typing was done using a multiplex PCR containing specific pairs of primers for SCCmec types I, II, III, IV (IVa, IVb, IVc and IVd) and V. Another multiplex PCR assay was used for the characterization of different types of ccr (Zhang et al. 2005).

Prophage typing

A multiplex PCR assay developed by Pantůček et al. (2004) was used for the characterization of SGA, SGB, SGF and its two serogroups SGFa and SGFb, SGL and SGD genes which in that order encode for hypothetical tail proteins, packaging proteins, hypothetical capsid proteins and major capsid proteins.

Detection of pvl gene

All CA-MRSA isolates were screened for pvl gene encoding for PVL toxin. PVL-specific primers were used as described by McClure et al. (2006). The PCR cycles were 10 min at 94 °C, 25 cycles of 45 s at 94 °C, 45 s at 55 °C, 75 s at 72 °C and a final extension step of 10 min at 72 °C.

Statistical analysis

Fisher's exact test was used to compare the significance of differences between the MRSA isolates in two STPs using GraphPad Prism version 5.0.

RESULTS

Identification of strains

A total of 576 S. aureus isolates were collected from both STPs, of which 364 (63%) were isolated from the incoming raw sewage, 124 (22%) from sludge and 88 (15%) were isolated from the outgoing-treated sewage of the STPs. Eighty (14%) isolates showed resistance to cefoxitin as a surrogate of oxacillin leaving 496 isolates as MSSA. Among the MSSA isolates, 245 (49%) were from the north STP and 251 (51%) were from the west STP (Table 1). The distribution of isolates varied according to the STPs, sampling locations and sampling dates (Table 1).

Table 1

The prevalence of MRSA and MSSA isolated from two STPs located in the north (N) and west (W) parts of Tehran during 2015 and 2016

STPDate of samplingIncoming sewage
Sludge
Outgoing sewage
Total
MSSAMRSAMSSAMRSAMSSAMRSAMSSAMRSA
N1 15 July 2015 41 (64%) 5 (63%) 13 (20%)**** 2 (25%)**** 10 (16%)**** 1 (12%)**** 64 
N2 15 October 2015 47(74%) 7(78%) 8(13%)**** 2(12%)**** 8(13%)**** 0 63 9 
N3 15 January 2016 35 (55%) 5 (63%) 17 (27%)**** 1 (12%)**** 12 (18%)**** 2 (25%)**** 64 
N4 15 April 2016 39 (63%) 4 (40%) 16 (26%)**** 3 (30%) 7 (11%)**** 3 (30%) 62 10 
W1 20 July 2015 43 (73%) 7 (54%) 9 (15%)**** 2 (15%)**** 7 (12%) **** 4 (31%)** 59 13 
W2 20 October 2015 37 (61%) 8 (73%) 11 (18%)**** 1 (9%)**** 13 (21%)**** 2 (18%)**** 61 11 
W3 20 January 2016 39 (63%) 5 (50%) 18 (29%)**** 2 (20%)**** 5 (8%)**** 3 (30%)** 62 10 
W4 20 April 2016 36(59%) 6(55%) 14(23%)**** 5(45%) 11(18%)**** 0 61 11 
Total 317 (64%) 47 (59%) 106 (21%) 18 (22%) 73 (15%) 15 (19%) 496 80 
STPDate of samplingIncoming sewage
Sludge
Outgoing sewage
Total
MSSAMRSAMSSAMRSAMSSAMRSAMSSAMRSA
N1 15 July 2015 41 (64%) 5 (63%) 13 (20%)**** 2 (25%)**** 10 (16%)**** 1 (12%)**** 64 
N2 15 October 2015 47(74%) 7(78%) 8(13%)**** 2(12%)**** 8(13%)**** 0 63 9 
N3 15 January 2016 35 (55%) 5 (63%) 17 (27%)**** 1 (12%)**** 12 (18%)**** 2 (25%)**** 64 
N4 15 April 2016 39 (63%) 4 (40%) 16 (26%)**** 3 (30%) 7 (11%)**** 3 (30%) 62 10 
W1 20 July 2015 43 (73%) 7 (54%) 9 (15%)**** 2 (15%)**** 7 (12%) **** 4 (31%)** 59 13 
W2 20 October 2015 37 (61%) 8 (73%) 11 (18%)**** 1 (9%)**** 13 (21%)**** 2 (18%)**** 61 11 
W3 20 January 2016 39 (63%) 5 (50%) 18 (29%)**** 2 (20%)**** 5 (8%)**** 3 (30%)** 62 10 
W4 20 April 2016 36(59%) 6(55%) 14(23%)**** 5(45%) 11(18%)**** 0 61 11 
Total 317 (64%) 47 (59%) 106 (21%) 18 (22%) 73 (15%) 15 (19%) 496 80 

**Significant at P ≤ 0.01,

****Significant at P < 0.0001.

Of the 80 MRSA isolates, 35 (44%) strains were from the north STP and 45 (56%) from the west STP. The distribution of these isolates in inlet, sludge and outlet samples was generally similar to that of MSSA isolates (Table 1). During the second round of sampling from the north STP in October 2015 and the fourth sampling from the west STP in April 2016, no MRSA was isolated from the outgoing sewage of both STPs which could be due to their low number in the incoming sewage to these STPs (Table 1, bold areas). The prevalence of both MRSA and MSSA isolates reduced during the treatment process in both STPs (Table 1).

Clonality of the MRSA strains

Biochemical fingerprinting with the PhPlate system of MRSA and MSSA isolates showed the presence of 21 PhP types [8 CTs and 13 single types (STs)] for MRSA strains (Supplementary Material, Figure 1) and 75 PhP types (48 CTs and 27 STs) for MSSA isolates.

Some of the CTs were found on different sampling occasions and in both STPs (Table 2).

Table 2

Clonality of the MRSA strains isolated from two STPs located in the north (N) and west (W) parts of Tehran during 2015 and 2016.

PhP typeNo.SiteSourceSCCmec typeccr typeProphage patternpvl gene
CT1 North Incoming III – 
West Incoming III – 
West Incoming III – 
West Sludge III – 
CT2 West Incoming III – 
West Sludge III – 
CT3 West Incoming III – 
West Sludge III – 
West Incoming III – 
10 West Outgoing III – 
11 West Sludge III – 
12 North Incoming III – 
13 West Incoming III – 
14 West Incoming III – 
15 West Incoming III – 
16 North Incoming III – 
17 North Incoming III – 
18 North Sludge III – 
19 North Sludge III – 
20 West Incoming III – 
21 West Incoming III – 
22 West Sludge III – 
23 West Incoming III – 
24 West Incoming III – 
25 West Sludge III – 
26 North Incoming III – 
27 North Incoming III – 
28 West Outgoing III – 
29 West Incoming III – 
30 North Incoming III – 
31 North Incoming III – 
32 North Outgoing III – 
33 West Outgoing III – 
34 North Incoming III – 
35 North Incoming III – 
36 West Incoming III – 
37 West Incoming III – 
CT4 38 West Outgoing III – 
39 North Incoming III – 
CT5 40 North Outgoing III – 
41 West Incoming III – 
CT6 42 West Sludge III – 
43 North Incoming III – 
44 North Sludge III – 
CT7 45 North Outgoing III – 
46 North Sludge III – 
47 North Sludge III – 
48 West Incoming III – 
49 West Outgoing III – 
50 North Incoming III – 
51 North Incoming III – 
52 North Outgoing III – 
53 North Sludge III – 
54 North Incoming III – 
55 West Incoming III – 
56 North Incoming III – 
57 North Incoming III – 
CT8 58 North Incoming III – 
59 North Sludge III – 
60 West Incoming III – 
61 West Outgoing III – 
62 West Sludge III – 
63 West Sludge III – 
64 West Incoming III – 
65 North Sludge III – 
66 North Outgoing III – 
67 North Outgoing III – 
ST1 68 West Incoming IVa 
ST2 69 West Incoming IVa 
ST3 70 North Incoming IVa 
ST4 71 North Incoming IVa 
ST5 72 West Incoming IVa 
ST6 73 West Incoming IVa 
ST7 74 West Incoming IVa 
ST8 75 West Incoming IVa 
ST9 76 North Incoming III – 
ST10 77 West Sludge IVa 
ST11 78 West Outgoing IVa 
ST12 79 West Outgoing III – 
ST13 80 West Outgoing III – 
PhP typeNo.SiteSourceSCCmec typeccr typeProphage patternpvl gene
CT1 North Incoming III – 
West Incoming III – 
West Incoming III – 
West Sludge III – 
CT2 West Incoming III – 
West Sludge III – 
CT3 West Incoming III – 
West Sludge III – 
West Incoming III – 
10 West Outgoing III – 
11 West Sludge III – 
12 North Incoming III – 
13 West Incoming III – 
14 West Incoming III – 
15 West Incoming III – 
16 North Incoming III – 
17 North Incoming III – 
18 North Sludge III – 
19 North Sludge III – 
20 West Incoming III – 
21 West Incoming III – 
22 West Sludge III – 
23 West Incoming III – 
24 West Incoming III – 
25 West Sludge III – 
26 North Incoming III – 
27 North Incoming III – 
28 West Outgoing III – 
29 West Incoming III – 
30 North Incoming III – 
31 North Incoming III – 
32 North Outgoing III – 
33 West Outgoing III – 
34 North Incoming III – 
35 North Incoming III – 
36 West Incoming III – 
37 West Incoming III – 
CT4 38 West Outgoing III – 
39 North Incoming III – 
CT5 40 North Outgoing III – 
41 West Incoming III – 
CT6 42 West Sludge III – 
43 North Incoming III – 
44 North Sludge III – 
CT7 45 North Outgoing III – 
46 North Sludge III – 
47 North Sludge III – 
48 West Incoming III – 
49 West Outgoing III – 
50 North Incoming III – 
51 North Incoming III – 
52 North Outgoing III – 
53 North Sludge III – 
54 North Incoming III – 
55 West Incoming III – 
56 North Incoming III – 
57 North Incoming III – 
CT8 58 North Incoming III – 
59 North Sludge III – 
60 West Incoming III – 
61 West Outgoing III – 
62 West Sludge III – 
63 West Sludge III – 
64 West Incoming III – 
65 North Sludge III – 
66 North Outgoing III – 
67 North Outgoing III – 
ST1 68 West Incoming IVa 
ST2 69 West Incoming IVa 
ST3 70 North Incoming IVa 
ST4 71 North Incoming IVa 
ST5 72 West Incoming IVa 
ST6 73 West Incoming IVa 
ST7 74 West Incoming IVa 
ST8 75 West Incoming IVa 
ST9 76 North Incoming III – 
ST10 77 West Sludge IVa 
ST11 78 West Outgoing IVa 
ST12 79 West Outgoing III – 
ST13 80 West Outgoing III – 

CT1–CT8: common types 1–8; ST1–ST13: single types 1–13.

The most CTs among MRSA strains, i.e. CT3 contained 31 (38.8%) isolates and was found in both STPs. This was followed by CT7 and CT8 containing 13 and 10 isolates, respectively (Table 2). Seventy (87.5%) MRSA isolates carried SCCmec type III and were PCR-positive with the ccrAB-α4-specific primers indicating the presence of type 3 ccr. Also, 10 (12.5%) isolates carried SCCmec type IVa with PCR band specific for type 2 ccr.

Sixty-seven MRSA isolates belonging to CT1–CT8, and also three STs (ST9, ST12 and ST13) were mecA-positive, carried SCCmec type III and harbored type 3 ccr. On the other hand, 10 STs (ST1–ST8, ST10 and ST11) were mecA- and pvl-positive and carried SCCmec type IV, type 2 ccr and SGA prophage type (Supplementary Material, Figures 2–4; Table 2).

Prophage typing

With exception of the Twort-like (SGD), we detected all types of prophages. SGA, SGB, SGL, SGF, SGFa and SGFb prophage genes were detected in 10 (12.5%), 26 (32.5%), 10 (12.5%), 80 (100%), 80 (100%) and 80 (100%) of the isolates, respectively. Based on prophage typing, three combinations of serogroups SGA, SGB, SGL, SGF and subgroup SGFa were identified among the isolates (Table 3). Prophage type 3, with SGF prophage type and its two subgroups, was the predominant pattern (67.5%; Table 3).

Table 3

Prophage patterns of the MRSA isolates tested and their frequency

Phage patternsPhage types
Frequency
SGASGBSGFSGFaSGFbSGL
+ + + + + + 10 (12.5%) 
– + + + + – 16 (20%) 
– – + + + – 54 (67.5%) 
Phage patternsPhage types
Frequency
SGASGBSGFSGFaSGFbSGL
+ + + + + + 10 (12.5%) 
– + + + + – 16 (20%) 
– – + + + – 54 (67.5%) 

Antibiotic susceptibility patterns

In all, 70 of the MRSA isolates and 309 MSSA isolates were multidrug-resistant ranging between 2 and 12 antibiotics, and although, 10 MRSA isolates showed resistance to only one antibiotic, 69 isolates were resistant to six or more antibiotics. In contrast, none of the MSSA isolates were resistant to more than six antibiotics tested (Table 4). The mean number of antibiotics to which MRSA were resistant (9.6 ± 1.5) was significantly (P < 0.0001) higher than that of MSSA isolates (4.1 ± 1.2). In both STPs, the number of isolates that were resistant to all 12 antibiotics decreased in samples collected from the incoming raw sewage to outgoing-treated sewage (P < 0.0001).

Table 4

Antibiotic resistance patterns among MRSA and MSSA strains isolated from two STPs in Tehran over 1 year

AntibioticsMRSA, n = 80MSSA, n = 496Total, n = 576
No resistance 5 (1) 5 (0.9) 
One antibiotic 10 (12.5) 182 (36.7) 192 (33.3) 
10 (12.5) 182 (36.7) 192 (33.3) 
Two antibiotics 42 (8.5) 42 (7.3) 
P, E 23 (4.7) 23 (4) 
P, CIP 19 (3.8) 19 (3.3) 
Three antibiotics 1 (1.25) 39 (7.9) 40 (6.9) 
P, CIP, TS 1 (1.25) 1 (0.1) 
P, CIP, E 5 (1) 5 (0.9) 
P, GM, MN 6 (1.2) 6 (1) 
P, RP, TS 9 (1.8) 9 (1.6) 
P, K, AN 11 (2.2) 11 (1.9) 
P, AN, MN 8 (1.6) 8 (1.4) 
Four antibiotics 91 (18.3) 91 (15.8) 
P, CIP, E, CD 13 (2.6) 13 (2.3) 
P, TS, AN, T 18 (3.6) 18 (3.1) 
P, E, K, CD 14 (2.8) 14 (2.4) 
P, CIP, T, RP 9 (1.8) 9 (1.6) 
P, TN, K, GM 7 (1.4) 7 (1.2) 
P, T, AN, RP 13 (2.6) 13 (2.3) 
P, TN, T, TS 9 (1.8) 9 (1.6) 
P, K, CD, MN 8 (1.6) 8 (1.4) 
Five antibiotics 109 (22) 109 (18.9) 
P, CIP, E, CD, K 5 (1) 5 (0.9) 
P, CIP, E, TN, RP 11 (2.2) 11 (1.9) 
P, TN, CD, TS, GM 15 (3) 15 (2.6) 
P, CIP, E, TN, T 14 (2.8) 14 (2.4) 
P, CIP, E, TN, K 12 (2.4) 12 (2.1) 
P, CIP, E, K, T 8 (1.6) 8 (1.4) 
P, CIP, E, GM, RP 6 (1.2) 6 (1) 
P, CIP, E, K, MN 9 (1.8) 9 (1.6) 
P, TN, K, AN, GM 11 (2.2) 11 (1.9) 
P, T, TS, GM, MN 6 (1.2) 6 (1) 
P, T, CD, TS, MN 8 (1.6) 8 (1.4) 
P, TN, CD, GM, MN 4 (0.8) 4 (0.7) 
Six antibiotics 1 (1.25) 28 (5.6) 29 (5) 
P, CIP, E, T, CD, RP 1 (1.25) 3 (0.6) 4 (0.7) 
P, CIP, E, K, AN, GM 7 (1.4) 7 (1.2) 
P, CIP, E, TN, K, AN 10 (2) 10 (1.7) 
P, CIP, E, AN, MN, GM 5 (1) 5 (0.9) 
P, E, CD, K, AN, GM 3 (0.6) 3 (0.5) 
Seven antibiotics 3 (3.75) 3 (0.5) 
P, CIP, E, T, CD, RP, MN 1 (1.25) 1 (0.1) 
P, CIP, T, K, TN, TS, RP 1 (1.25) 1 (0.1) 
P, CIP, T, K, TN, RP, MN 1 (1.25) 1 (0.1) 
Eight antibiotics 8 (10) 8 (1.4) 
P, CIP, E, CD, TS, RP, K, TN 1 (1.25) 1 (0.1) 
P, CIP, E, T, CD, K, TN, TS 1 (1.25) 1 (0.1) 
P, CIP, E, CD, K, AN, TN, RP 5 (6.25) 5 (0.9) 
P, CIP, T, K, AN, TN, RP, GM 1 (1.25) 1 (0.1) 
Nine antibiotics 15 (18.75) 15 (2.6) 
P, CIP, E, T, CD, K, AN, TN, RP 7 (8.75) 7 (1.2) 
P, CIP, E, CD, K, AN, TN, TS, RP 1 (1.25) 1 (0.1) 
P, CIP, E, CD, K, AN, TN, RP, GM 2 (2.5) 2 (0.3) 
P, CIP, E, T, K, AN, TN, TS, GM 4 (5) 4 (0.7) 
P, CIP, E, T, K, AN, TN, TS, MN 1 (1.25) 1 (0.1) 
Ten antibiotics 19 (23.75) 19 (3.3) 
P, CIP, E, T, CD, K, AN, TN, TS, RP 2 (2.5) 2 (0.3) 
P, CIP, E, T, CD, K, AN, TN, TS, MN 1 (1.25) 1 (0.1) 
P, CIP, E, T, CD, K, AN, TN, TS, GM 2 (2.5) 2 (0.3) 
P, CIP, E, T, CD, K, AN, TN, RP, MN 7 (8.75) 7 (1.2) 
P, CIP, E, T, CD, K, TN, TS, RP, MN 3 (3.75) 3 (0.5) 
P, CIP, E, CD, K, AN, TN, TS, RP, GM 2 (2.5) 2 (0.3) 
P, CIP, E, T, K, AN, TN, TS, MN, GM 2 (2.5) 2 (0.3) 
Eleven antibiotics 21 (26.25) 21 (3.6) 
P, CIP, E, T, CD, K, AN, TN, TS, RP, GM 6 (7.5) 6 (1) 
P, CIP, E, T, CD, K, AN, TN, TS, RP, MN 3 (3.75) 3 (0.5) 
P, CIP, E, T, CD, K, AN, TN, TS, MN, GM 9 (11.25) 9 (1.6) 
P, CIP, E, T, CD, K, AN, TN, RP, MN, GM 3 (3.75) 3 (0.5) 
Twelve antibiotics 2 (2.5) 2 (0.3) 
P, CIP, E, T, CD, K, AN, TN, TS, RP, MN, GM 2 (2.5) 2 (0.3) 
AntibioticsMRSA, n = 80MSSA, n = 496Total, n = 576
No resistance 5 (1) 5 (0.9) 
One antibiotic 10 (12.5) 182 (36.7) 192 (33.3) 
10 (12.5) 182 (36.7) 192 (33.3) 
Two antibiotics 42 (8.5) 42 (7.3) 
P, E 23 (4.7) 23 (4) 
P, CIP 19 (3.8) 19 (3.3) 
Three antibiotics 1 (1.25) 39 (7.9) 40 (6.9) 
P, CIP, TS 1 (1.25) 1 (0.1) 
P, CIP, E 5 (1) 5 (0.9) 
P, GM, MN 6 (1.2) 6 (1) 
P, RP, TS 9 (1.8) 9 (1.6) 
P, K, AN 11 (2.2) 11 (1.9) 
P, AN, MN 8 (1.6) 8 (1.4) 
Four antibiotics 91 (18.3) 91 (15.8) 
P, CIP, E, CD 13 (2.6) 13 (2.3) 
P, TS, AN, T 18 (3.6) 18 (3.1) 
P, E, K, CD 14 (2.8) 14 (2.4) 
P, CIP, T, RP 9 (1.8) 9 (1.6) 
P, TN, K, GM 7 (1.4) 7 (1.2) 
P, T, AN, RP 13 (2.6) 13 (2.3) 
P, TN, T, TS 9 (1.8) 9 (1.6) 
P, K, CD, MN 8 (1.6) 8 (1.4) 
Five antibiotics 109 (22) 109 (18.9) 
P, CIP, E, CD, K 5 (1) 5 (0.9) 
P, CIP, E, TN, RP 11 (2.2) 11 (1.9) 
P, TN, CD, TS, GM 15 (3) 15 (2.6) 
P, CIP, E, TN, T 14 (2.8) 14 (2.4) 
P, CIP, E, TN, K 12 (2.4) 12 (2.1) 
P, CIP, E, K, T 8 (1.6) 8 (1.4) 
P, CIP, E, GM, RP 6 (1.2) 6 (1) 
P, CIP, E, K, MN 9 (1.8) 9 (1.6) 
P, TN, K, AN, GM 11 (2.2) 11 (1.9) 
P, T, TS, GM, MN 6 (1.2) 6 (1) 
P, T, CD, TS, MN 8 (1.6) 8 (1.4) 
P, TN, CD, GM, MN 4 (0.8) 4 (0.7) 
Six antibiotics 1 (1.25) 28 (5.6) 29 (5) 
P, CIP, E, T, CD, RP 1 (1.25) 3 (0.6) 4 (0.7) 
P, CIP, E, K, AN, GM 7 (1.4) 7 (1.2) 
P, CIP, E, TN, K, AN 10 (2) 10 (1.7) 
P, CIP, E, AN, MN, GM 5 (1) 5 (0.9) 
P, E, CD, K, AN, GM 3 (0.6) 3 (0.5) 
Seven antibiotics 3 (3.75) 3 (0.5) 
P, CIP, E, T, CD, RP, MN 1 (1.25) 1 (0.1) 
P, CIP, T, K, TN, TS, RP 1 (1.25) 1 (0.1) 
P, CIP, T, K, TN, RP, MN 1 (1.25) 1 (0.1) 
Eight antibiotics 8 (10) 8 (1.4) 
P, CIP, E, CD, TS, RP, K, TN 1 (1.25) 1 (0.1) 
P, CIP, E, T, CD, K, TN, TS 1 (1.25) 1 (0.1) 
P, CIP, E, CD, K, AN, TN, RP 5 (6.25) 5 (0.9) 
P, CIP, T, K, AN, TN, RP, GM 1 (1.25) 1 (0.1) 
Nine antibiotics 15 (18.75) 15 (2.6) 
P, CIP, E, T, CD, K, AN, TN, RP 7 (8.75) 7 (1.2) 
P, CIP, E, CD, K, AN, TN, TS, RP 1 (1.25) 1 (0.1) 
P, CIP, E, CD, K, AN, TN, RP, GM 2 (2.5) 2 (0.3) 
P, CIP, E, T, K, AN, TN, TS, GM 4 (5) 4 (0.7) 
P, CIP, E, T, K, AN, TN, TS, MN 1 (1.25) 1 (0.1) 
Ten antibiotics 19 (23.75) 19 (3.3) 
P, CIP, E, T, CD, K, AN, TN, TS, RP 2 (2.5) 2 (0.3) 
P, CIP, E, T, CD, K, AN, TN, TS, MN 1 (1.25) 1 (0.1) 
P, CIP, E, T, CD, K, AN, TN, TS, GM 2 (2.5) 2 (0.3) 
P, CIP, E, T, CD, K, AN, TN, RP, MN 7 (8.75) 7 (1.2) 
P, CIP, E, T, CD, K, TN, TS, RP, MN 3 (3.75) 3 (0.5) 
P, CIP, E, CD, K, AN, TN, TS, RP, GM 2 (2.5) 2 (0.3) 
P, CIP, E, T, K, AN, TN, TS, MN, GM 2 (2.5) 2 (0.3) 
Eleven antibiotics 21 (26.25) 21 (3.6) 
P, CIP, E, T, CD, K, AN, TN, TS, RP, GM 6 (7.5) 6 (1) 
P, CIP, E, T, CD, K, AN, TN, TS, RP, MN 3 (3.75) 3 (0.5) 
P, CIP, E, T, CD, K, AN, TN, TS, MN, GM 9 (11.25) 9 (1.6) 
P, CIP, E, T, CD, K, AN, TN, RP, MN, GM 3 (3.75) 3 (0.5) 
Twelve antibiotics 2 (2.5) 2 (0.3) 
P, CIP, E, T, CD, K, AN, TN, TS, RP, MN, GM 2 (2.5) 2 (0.3) 

MN, minocycline; TS, sulfamethoxazole-trimethoprim; GM, gentamicin; RP, rifampin; CD, clindamycin; TN, tobramycin; T, tetracycline; K, kanamycin; AN, amikacin; E, erythromycin; CIP, ciprofloxacin; PG, penicillin G.

The MIC of the MRSA strains was determined using the E-test. The results showed that all isolates had MIC ≥ 4 μg/ml to oxacillin. Also, 54% (n = 19) and 51% (n = 23) of MRSA isolates isolated from north and west STPs, respectively, showed a high level of resistance (MIC ≥ 256 μg/ml) to oxacillin (Figure 1). In contrast, 9% (n = 3) and 16% (n = 7) of strains isolated from north and west STPs, respectively, had a low level of resistance (MIC ≥ 4 μg/ml). The MIC of 35% of the remaining isolates varied from 24 to 128 μg/ml. The frequency of MRSA strains showing resistance to 256 μg/ml of oxacillin was decreased during the process of sewage treatment (P < 0.0001 and P = 0.0006 in north and west STPs, respectively). In contrast, the number of isolates with low MIC to oxacillin was increased in the process.

Figure 1

MICs range to oxacillin among MRSA strains: MRSA had MIC ≥4 μg/ml. *Significant at P ≤ 0.05, ***Significant at P ≤ 0.001, ****Significant at P < 0.0001.

Figure 1

MICs range to oxacillin among MRSA strains: MRSA had MIC ≥4 μg/ml. *Significant at P ≤ 0.05, ***Significant at P ≤ 0.001, ****Significant at P < 0.0001.

DISCUSSION

Very little is known about the prevalence and persistence of antibiotic resistance S. aureus and, in particular, MRSA populations in the sewage and wastewater in Iran, and to the best of our knowledge, this is the first report of the detection of MRSA at municipal STPs in Iran. The presence of MRSA strains in wastewater treatment plants (WWTPs) has been reported in different countries (Schwartz et al. 2003; Volkmann et al. 2004; Börjesson et al. 2010; Goldstein et al. 2012). In the present study, 14% of the S. aureus strains isolated were MRSA and they were found in 22 of 24 (92%) samples taken from different sites of the STPs. While MRSA were present in 100% of incoming raw samples, only six of eight (75%) effluent samples were MRSA-positive, indicating that they were killed during the STP process. It was rather difficult to compare the proportion of the MRSA found in our study with those reported elsewhere due to differences in methodology and the media used. For instance, Börjesson et al. (2010) used Brilliance MRSA Agar and MRSASelect Chromogenic Agar for the isolation of MRSA and isolated 189 MRSA strains from WWTPs. In another study, these researchers showed that the concentration of mecA gene decreased from incoming raw to outgoing samples during the sewage treatment process (Börjesson et al. 2009b). Volkmann et al. (2004) employed a TaqMan-based real-time PCR method for the detection of MRSA isolates carrying mecA gene in municipal and clinical wastewater and showed that mecA gene was only detected among MRSA isolates with clinical wastewater origin. In another study from Germany (Schwartz et al. 2003), mecA gene was only common among biofilms in hospital wastewater, and none of the municipal wastewater, surface water and drinking water biofilms were positive for mecA resistance gene. Furthermore, to assess the persistence of staphylococci in hospital biofilms, samples were also cultured on synthetic medium, and none of the S. aureus isolates showed to be resistant to oxacillin.

We previously reported the presence of MRSA and MSSA strains among patients with CA, HA, meat samples and poultry farms (Rahimi et al. 2014; Rahimi & Karimi 2015; Rahimi & Shokoohizadeh 2016; Rahimi & Shafiei 2019). Most of these strains belonged to few CTs. We compared CTs of MRSA and MSSA strains isolated in this study with those saved from previous studies done in our group (Javidnia et al. 2013; Rahimi et al. 2014; Rahimi & Shokoohizadeh 2016; Rahimi & Shafiei 2019) and found that some of the CTs with identical SCCmec and prophage types were common in all samples (data not shown). We also found diverse PhP types of both MSSA and MRSA in the STPs over 1 year of sampling.

STPs can serve as an important reservoir for the dissemination of multidrug-resistant strains of S. aureus and, in particular, MRSA in communities. In the present study, despite the variation in the number of S. aureus found between different samples in both STPs, we found the presence of few CTs of MRSA with a similar antibiotic resistance pattern (data not shown). Some of these CTs also had identical SCCmec, ccr types and prophage types found in most samples, indicating that these strains either had common sources or persisted in the STPs or a combination of both. It must be noted, however, that there was a marked reduction in the number of MSSA and MRSA isolates (showed high-level resistance to oxacillin) recovered from the outlet samples as opposed to inlet samples of both STPs. These data indicated that the sewage treatment process, in general, is useful for removing most S. aureus populations, but some strains (showed low-level resistance to oxacillin) may have a better ability to survive the STP process. Similar results have been found by Thompson et al. (2013) who showed that certain clonal types of MRSA can survive transmission from hospital to STP via the sewer system and resist the STP treatment process. In our study, some clones of MRSA strains that survived the treatment process in each STP showed resistance to most antibiotics tested; however, some harbored different classes of prophages and different virulence factors.

We also found a decrease in MRSA populations in samples collected from the incoming to the outgoing effluent of the STPs. Similar results have also been found by Goldstein et al. (2012) who showed a major reduction in the population of MRSA isolates in the anaerobic step of the sewage treatment process with no MRSA isolates detected in the outlet samples. Despite these findings, the presence of MRSA strains and mecA gene has been reported in both raw and treated sewages in other studies (Naquin et al. 2015; Boopathy 2017). Whether this is due to the difference between certain clones of MRSA or due to the inadequate process of some STPs remains to be identified.

The SCCmec types III and IV were found in highest frequencies among the MRSA strains. We previously found similar results among clinical (Rahimi et al. 2014, 2016; Rahimi & Shokoohizadeh 2016) and animal samples (Rahimi & Shafiei 2019) in Iran. These findings are different from a study in the USA which showed that most of the MRSA strains in STPs contained SCCmec types IV and II, respectively (Goldstein et al. 2012). It seems that there is no significant association between MRSA survival and SCCmec types as shown by Levin-Edens et al. (2011). On the other hand, Börjesson et al. (2010) showed that the MRSA isolates harbored SCCmec type IV were able to survive for a longer period time in the environment than other SCCmec types.

It should be noted that carriage of SCCmec IV by the MRSA strains does not impose an energetic cost, and also SCCmec type IV-positive MRSA strains only show resistance to β-lactam antibiotics and have the ability to acquire new resistance genes via the horizontal gene transfer process.

The PhP typing method used in this study is a powerful method specifically developed for typing of Staphylococci (Jung et al. 1995; Björkqvist et al. 2002; Thompson et al. 2013; Rahimi et al. 2014; Rahimi & Shokoohizadeh 2016). For examples, Rahimi et al. (2014, 2016) reported the presence of 33 PhP types (consisting of 18 CTs and 15 STs) and 33 PhP types (consisting of 29 CTs and 4 STs) among MRSA strains in Tehran, respectively. The system has been used in many epidemiological studies alone or in combination with molecular methods (Björkqvist et al. 2002; Nilsdotter-Augustinsson et al. 2007; Javidnia et al. 2013). The results of PhP typing of S. aureus isolates showed the presence of different CTs of MSSA and MRSA in both STPs, indicating their widespread dissemination within the effluent entering these STPs. Interestingly, the dominant CTs of MRSA, i.e. CT3 that was present in samples from both STPs, harbored two different prophage types and subtypes, suggesting independent evolution of this clone in the community. Different antibiotic resistance and prophage patterns were also seen within each CTs. However, strains with the same PhP type had identical prophage and antibiotic resistance pattern and were isolated from different samples in each STP (i.e. CT6 and CT7), further emphasizing the persistence of specific clones in sewage over a year period of this study.

STPs are one of the major sources of genetic exchange, especially antibiotic (e.g., tetracycline, erythromycin and vancomycin) resistance genes among bacteria (Tao et al. 2016; Barancheshme & Munir 2018). They have shown the positive correlation between antibiotic resistance genes (ARGs) and their correlation with antibiotics in the STPs and also revealed that the high density of bacteria and treatment process could create an ideal environment for ARG exchange. It was therefore not surprising that within some of the CTs, there existed more than one antibiotic resistance pattern. Moreover, similar to other reports from Iran, none of the strains showed resistance to vancomycin, linezolid or quinupristin-dalfopristin which could be due to restricted use of these antibiotics in this country (Fatholahzadeh et al. 2008; Goudarzi et al. 2016; Rahimi & Shafiei 2019). Different patterns of antibiotic resistance were observed among the isolates; however, compared to MSSA strains, a high number of MRSA were resistant to six or more antibiotics (5.6 versus 86.2%, respectively, P < 0.0001). This could be partly due to the high prevalence of multidrug-resistant MRSA strains in hospitals and the communities in Tehran (Javidnia et al. 2013; Goudarzi et al. 2016) which find their ways to STPs.

In this study, three different prophage patterns were detected among our strains. The presence of different prophage patterns has been reported previously in Iran and other countries (Pantůček et al. 2004; Workman et al. 2006; Rahimi et al. 2014). Moreover, we found that strains lacking SGA prophage type (prophage patterns 2 and 3) mainly belonged to CTs, suggesting that the former prophage pattern exists in small numbers in the community and can only be detected in STPs where samples from wide sources are received. Interestingly, compared to SGA-positive MRSA strains that showed susceptibility to all classes of antibiosis tested in this study (except for penicillin), all SGA negative strains were resistant to 1–12 antibiotics (data not shown). Whether there is a genetic linkage between the SGA prophage type and the antibiotics resistance genes is yet to be identified, but this finding suggests that STPs can be valuable sources for detecting minor clones of antibiotic-resistant bacteria that may, if they disseminate in the community, cause a major health problem.

In conclusion, our findings suggest that multiresistant MRSA strains are commonly present in STPs in Tehran, Iran. These strains were found in samples collected from the incoming effluent, sludge and outgoing-treated samples indicating their survival in STPs. The fact that identical MRSA strains were found at different sampling occasions in the same STP suggests that either these strains were entering the sewage system from common source(s) or they have the ability to survive in the raw sewage or a combination of both. The high prevalence of MRSA strains with SCCmec type IV and different prophage types and antibiotic resistance in treated STP wastes suggest a potential risk to the public in this country.

ACKNOWLEDGEMENTS

This research was funded by a grant from the Ministry of Health of Iran, Deputy of Research and Innovation.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest with the organization that sponsored this research and publications arising from this research.

DATA AVAILABILITY STATEMENT

All relevant data are included in the paper or its Supplementary Information.

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