Occurrence of Staphylococcus spp. in the wastewaters from Iran: Diversity, antimicrobial resistance, and virulence potential

Wastewater is a complex ecological environment hosting different types of microorganisms (Börjesson et al. 2010). Pathogenic bacteria enter the wastewater from hospitals, or from any diseased people or healthy carriers. Animal wastes often originate from farms, slaughterhouses, meat processing industries, and from rodents found around sewages (Dweba et al. 2018). When untreated wastewater reaches water used for drinking or irrigation of vegetable farms there can be significant public health risks. Pathogens in wastewater are transmissible through ingesting drinking water or crops contaminated with sewage or as a result of contact with the animal, human, or insect carriers (Wagner & Loy 2002). The wastewater is an environmental reservoir which plays a significant role in the development and spread of antimicrobial resistance (Martinez-Huitle & Ferro 2006; Börjesson et al. 2010). Several studies showed the residues of many antibiotics in wastewaters around the world (Watkinson et al. 2009). Residual antibiotics are capable of selecting resistant bacteria through inhibiting or killing the susceptible organisms. Resistant bacteria survive in the presence of antibiotics and act as vectors for the dissemination of antibiotic-resistance genes (Kruse et al. 1999). Excessive and uncontrolled application of antibiotics in human medicine, veterinary, and as growth promoting agents in food-producing animals lead to an increment in antibiotic resistance and in the spread of bacteria carrying resistance genes (Iversen et al. 2002; Dweba et al. 2018). The major risk threatening general health is the environmental bacteria transferring resistance encoding genes to human pathogens (Moges et al. 2014). The majority of studies regarding the characterization of resistant bacteria in wastewater have emphasized the fecal pollution indicator bacteria (Zaatout et al. 2021). However, other antibiotic-resistant clinically significant pathogens have been reported in wastewater as well (Nishiyama et al. 2021).

Staphylococcus spp. are normal flora, found in mucus membranes and skin of human and other mammals (Gómez et al. 2016) which are capable to enter hospital, municipal, livestock, and poultry wastewater. Hence, they have been frequently isolated from wastewater of various sources (Faria et al. 2009; Börjesson et al. 2010; Goldstein et al. 2012; Heß & Gallert 2014; Kumar et al. 2015). Staphylococcus genus comprises several species classified into coagulase-positive Staphylococci (CoPS) and coagulase-negative Staphylococci (CoNS) groups. Most staphylococcal infections are caused by CoPS. So far, seven CoPS species have been recognized: Staphylococcus intermedius, Staphylococcus aureus, Staphylococcus schleiferi subsp. coagulanse, Staphylococcus delphini, Staphylococcus hyicus, Staphylococcus lutrae, and Staphylococcus pseudintermedius. S. aureus is the most common and highly virulent species in the genus responsible for a diverse array of life-threatening nosocomial and community-acquired infections (Sasaki et al. 2010; Holmes et al. 2014; Kumar et al. 2015). Instead, CoNS established themselves as opportunistic pathogens which are less virulent than CoPS. However, they can cause clinically significant infections in immunocompromised people and in those with implanted foreign materials such as catheters, shunts, and prosthetic joints (Hitzenbichler et al. 2017). There are currently over 40 identified species in the CoNS group, in which Staphylococcus haemolyticus, Staphylococcus epidermidis, and Staphylococcus saprophyticus are mostly responsible for infection in human beings (Hitzenbichler et al. 2017). Other species, such as Staphylococcus hominis, Staphylococcus warneri, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus cohnii, Staphylococcus xylosus, Staphylococcus saccharolyticus, and Staphylococcus lugdunensis are sometimes collected from clinical specimens (Garza-González et al. 2010). The most striking situation regarding Staphylococci is emerging methicillin-resistant strains. Methicillin is the first semisynthetic penicillinase-stable penicillins including oxacillin, cloxacillin, naficillin, and dicloxacillin. Staphylococcus spp. resistant to this group are historically termed Methicillin-Resistant Staphylococci (MRS). What sets it apart is that MRS isolates are resistant to all other currently available β-lactam antimicrobial agents, with the exception of ceftaroline (CLSI 2017). Additionally, MRS isolates are commonly resistant to several other classes of antibiotics such as aminoglycosides, chloramphenicol, quinolones, macrolides, and tetracycline (Kumar et al. 2015). The resistance against methicillin is mainly mediated by mecA gene. Staphylococcal cassette chromosome mec (SCC mec) has been reported as the only vector for the mec A gene (Zhang et al. 2005).

Studies showed large differences in Staphylococci population structure among wastewaters. The structure of the bacterial community is likely influenced by the operating parameters and composition of the effluents (Börjesson et al. 2010). Some studies have shown that common disinfecting agents such as chlorine and ultra-violet irradiation could not efficiently reduce the antibiotic-resistant bacteria and antibiotic resistance genes in the wastewater disinfecting process (Munir et al. 2011). So, understanding the microbial communities is essential for the implementation of effective disinfection approaches in wastewater treatment facilities (Gonzalez-Martinez et al. 2018).

The goals of the current study are (i) to assess the occurrence of Staphylococcus spp. in hospitals, slaughterhouses (poultry and livestock), and municipal wastewater sources in Iran; (ii) to evaluate the antimicrobial resistance profile of the isolates against common antibiotics; (iii) to investigate the occurrence of Methicillin-Resistant S. aureus (MRSA) isolates; (iv) to identify the SCCmec- and spa type of S. aureus isolates; (v) to study the genetic background responsible for aminoglycoside and macrolide resistance in S. aureus isolates; and (vi) to identify the virulence determinants of S. aureus isolates.

Sampling was done from wastewaters from municipal, hospitals, and slaughterhouses (poultry and livestock) in Ardabil, northwestern Iran. From August 2017 to June 2018, a total of 40 non-treated wastewater samples were taken twice a month on the 1st and 15th of each month. Specimens were collected in sterile glass bottles (500 mL), and transferred to the microbiology laboratory in ice cold containers, and were analyzed in less than 6 h after collection (Rahimi & Bouzari 2015; Gómez et al. 2016). This study was approved by the regional ethics committee of Islamic Azad University of Shiraz (IR.IAU.SHIRAZ.REC.1399.014).

Samples (250 mL) were diluted 10-fold with 0.9% NaCl (normal saline) and filtered through a 0.45 μm pore membrane (Millipore Corporation, Bedford, MA, USA). For enrichment of Staphylococcus isolates captured on filters, the filters were placed in tubs containing 40 mL M Staphylococcus broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), shaken and incubated at 37 °C for 24 h. A 10-μL aliquot of overnight bacterial culture was transferred on Mannitol salt agar (BioMaxima, Lublin, Poland) for the isolation of total Staphylococcus spp. Then, the plates were incubated at 37 °C for 24 h (Goldstein et al. 2012). Up to eight colonies per wastewater specimen with morphology resembling Staphylococci were cultured on Brain Heart Infusion (BHI) agar (BioMaxima, Poland) to provide a richer environment for bacterial growth, and then initially identified by conventional microbiological tests [Catalase, Gram stain, DNase (Merck, Darmstadt, Germany) and tube coagulase tests]. CoNS were identified using a commercial kit (Microgen™ Staph-ID system, UK). This system employs 12 standardized biochemical tests to characterize important species of the Staphylococci. S. aureus isolates were identified by PCR targeting the thermonuclease gene (nuc) with the primers and cycling conditions shown in Supplementary material, Table S1 (Murakami et al. 1991). PCR was carried out in a total volume of 25 μL using 12.5 μL of Premix Taq^® mix (CinnaGen, Tehran, Iran), 1 μL of template DNA (5 μg), l μL (10 pmol) of each forward and reverse primers, and 9.5 μL of nuclease-free water. PCR products were analyzed via electrophoresis at 100 V for 1 h in a 1% agarose gel (Sinaclon, Tehran, Iran), stained with DNA-safe stain (Sinaclon, Tehran, Iran) and DNA bands were visualized using UV illumination (Uvi Tec, Cambridge, UK). S. aureus ATCC 33591 was used as the positive control and the negative control was nuclease-free distilled water.

After identification of the isolates, two purified colonies grown on BHI agar were transferred into 10 mL of Trypticase soy broth (TSB) (Merck, Germany) and incubated overnight at 37 °C. Then, the cultures were aliquoted in 1.5 mL of cryovials including 15% glycerol (Merck, Germany), and stored at −80 °C until further use.

Antimicrobial susceptibility analysis was performed with the disk diffusion approach on Muller–Hinton agar (BioMaxima, Poland) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2017). The evaluated antibiotics (Padtan Teb, Iran) were chloramphenicol (30 μg), co-amoxiclav (30 μg), tetracycline (30 μg), penicillin (10 μg), erythromycin (15 μg), ciprofloxacin (10 μg), cefazolin (30 μg), clindamycin (2 μg), imipenem (10 μg), ceftriaxone (100 μg), rifampicin (30 μg), mupirocin (5 μg), trimethoprim sulfamethoxazole (25 μg), azithromycin (15 μg), gentamicin (10 μg), and amikacin (30 μg).

Methicillin resistance was investigated using the cefoxitin disk diffusion test (inhibition zone diameter ≤21 mm indicated MRSA) and oxacillin minimum inhibitory concentration (MIC) (≥4 μg/mL indicated MRSA) were determined using the agar dilution method (concentration range: 0.125–512 μg/mL) (CLSI 2017).

MICs of erythromycin, kanamycin, and tobramycin were assessed using a standard agar dilution technique (0.125–512 μg/mL). The isolates with MICs of ≥8 (μg/mL), ≥64 (μg/mL) and ≥16 (μg/mL) were defined as erythromycin-, kanamycin- and tobramycin-resistant, respectively (CLSI 2017).

BHI agar including 6 μg/mL vancomycin (Bio Basic, Canada) was used to screen isolates resistant to vancomycin. The MICs for isolates growing on BHI–vancomycin screening agar were determined by agar dilution technique (0.125–512 μg/mL). Resistance against vancomycin was considered as MIC ≥ 16 (μg/mL). The susceptibility testing was carried out and interpreted based on the CLSI's guidelines. S. aureus ATCC33591 was used as a quality control strain (CLSI 2017).

The D-test was carried out to identify inducible clindamycin resistance in clindamycin-susceptible staphylococcal isolates resistant to erythromycin. The clindamycin disks (2 μg) were located 25 mm apart (center to center) from erythromycin disks (15 μg). Following incubation at 37 °C for 18 h, the D phenotype was indicated by bacterial lawn exhibiting flattening of the inhibition zone (D formation) surrounding clindamycin disks near the erythromycin disks (Steward et al. 2005; Heß & Gallert 2014; CLSI 2017).

Erythromycin-resistant genes (erm A, erm C, erm B, erm TR, msr A), aminoglycoside-resistant genes (ant, aac, aph(2)-Ib, aph (2)-Ic, aph(2)-Id), and methicillin-resistant gene (mec A) were detected through PCR using specific primers (Supplementary material, Table S1) (Choi et al. 2003; O'Sullivan et al. 2006; Leelaporn et al. 2008; Dibah et al. 2014). DNA from previously identified isolates containing target genes was used as a positive control in the PCR experiment (Omid et al. 2021).

PCR (Supplementary material, Table S1) was used to detect the genes encoding enterotoxins (sea, seb, sec, and sed), exfoliative toxins (eta and etb), hemolysin toxins (hla and hld), Panton-Valentine leucocidin (PVL,lukF/S), and the toxic-shock syndrome toxin (tst) (Omid et al. 2021). DNA from previously identified isolates carrying the corresponding virulence genes was used as a positive control in PCR experiments (Omid et al. 2021).

SCCmec type was determined on all mecA-positive isolates. Two series of multiples PCR assays were used to detect SCCmec types and subtypes I, II, III, IV_a, IV_b, IV_c, IV_d, and V according to the conditions in Supplementary material, Table S1 with some changes in the PCR cycling conditions and annealing temperature (Omid et al. 2021). Amplicons were analyzed as introduced previously in this paper. DNA harvested from previously identified isolates with the known SCCmec types was used as a positive control in PCR testing (Omid et al. 2021).

The S. aureus strains' genetic profiles were generated using protein A (spa) typing. The spa gene polymorphic X region was amplified via primers and PCR conditions reported in Supplementary material, Table S1 as introduced by Harmsen et al. (2003). Both strands of the amplified fragments were sequenced by Macrogen (South Korea). The spa types were identified using the spa typing website available online at http://www.spaserver.ridom.de.

The susceptibility profile of staphylococcal isolates, evaluated through the disk diffusion assay, is provided in Tables 1 and 2. In general, imipenem, rifampicin, mupirocin, vancomycin (for each, n = 0/69; 0%), and penicillin (n = 49/69; 89%) were the antibiotics with the highest and the lowest activity against CoPS isolates, respectively. CoNS isolates were the most resistant to erythromycin (n = 47/86; 55%) followed by tetracycline (n = 42/86; 49%) and azithromycin (n = 29/86; 34%), respectively.

As shown in Supplementary material, Table S2, totally, 49% (n = 34/69) CoPS isolates were multidrug-resistant (MDR) (resistant against three or more classes of antibiotics). Among CoPS species, S. aureus (n = 28/55; 51%), S. intermedius (n = 4/10; 40%), and S. hyicus (n = 2/3; 67%) showed an MDR phenotype, respectively.

Antibiotic resistance patterns of CoNS isolates are shown in Supplementary material, Table S3. Results showed that in total, 46.5% (n = 40/86) of CoNS isolates were MDR. Among CoNS species, 55.5% (n = 5/9), 58% (n = 11/19), 75% (n = 3/4), 62.5% (n = 5/8), 44% (n = 14/32), 17% (n = 1/6), and 33% (n = 1/3) of S. chromogenes, S. lentus, S. simulanse, S. warneri, S. xylosus, S. hominis, and S. cohnii subsp. urealyticum showed an MDR phenotype, respectively.

According to oxacillin MIC (Supplementary material, Table S4) and mecA PCR testing, 2% (n = 1/55) of S. aureus isolates was identified as MRSA. This isolate belonged to SCCmec type V_.

According to MIC testing, resistance to aminoglycoside antibiotics was observed in 11% (n = 6/55), 4% (n = 2/55), and 2% (n = 1/55) of S. aureus isolates against tobramycin, kanamycin, and gentamycin, respectively. The resistance gene aac was detected in 75% (n = 9/12) and the ant and aphc genes were identified in 33% (for each, n = 4/12) of aminoglycoside-resistant isolates. Other aminoglycoside resistance-encoding genes were not identified in our S. aureus isolates (Figure 3). A total of four different genetic profiles (aac, ant, aac/aphc, aac/ant/aphc) were obtained for aminoglycoside resistance-encoding genes with profile only aac having the highest (42%) frequency (Supplementary material, Table S5).

In total, among eight S. aureus isolates subjected to spa typing, six different spa types were identified and one (12.5%) isolate was not typeable. spa type t346 was detected in 25% (n = 2) and types t026, t14870, t937, t19215, and t17068 were detected in 12.5% (for each, n = 1) of S. aureus isolates, respectively. The t19215 is a new spa type identified in this study (spa.ridom.de/194598, 2020, MRSA).

Despite the extensive investigation on Staphylococcus spp. in clinical settings, systematic assessments of their role in the aquatic environment and sewage samples are limited in most parts of the world. We found that the dominant species of Staphylococcus spp. was CoNS (55.5%) which itself is dominated by in wastewater samples. Similar results were reported from Spain (75%), Germany (57.3%), and Portugal (78.7%) previously (Faria et al. 2009; Heß & Gallert 2014; Gómez et al. 2016). CoNS species was dominated by S. xylosus (37%) among 10 different types identified in our study, while S. saprophyticus was reported as a dominant CoNS species type in wastewater in Europe (Faria et al. 2009; Heß & Gallert 2014). S. saprophyticus has been associated with urinary tract infections in young women (Jordan et al. 1980). Other CoNS species, with low frequencies, found in the current study were S. epidermidis, S. hominis, and S. haemolyticus, which are common pathogens responsible for human and veterinary diseases (Nagase et al. 2002). Similar results were reported around the world on the low occurrence of these species in wastewater. The prevalence was estimated to be ≤6.2% for S. hominis and S. haemolyticus and 23% for S. epidermidis in wastewater (Faria et al. 2009; Heß & Gallert 2014; Gómez et al. 2016).

CoNS are among the animals and human normal microbiota (Nagase et al. 2002) entering sewage through animal and human excrements (Aarestrup 2001; Werckenthin et al. 2001; Heß & Gallert 2014). Therefore, the variation in the occurrence of CoNS in wastewaters may arise from the variation in the abundance of microorganisms in animal and human host bodies at different regions.

In the present study, 44.5% of the isolates were characterized as CoPS, majorly belonging to S. aureus (80%). Most of the S. aureus isolates (67%) were found in hospital wastewater. Our findings are in contrast with earlier studies indicating that S. aureus is absent or less prevalent (0.4–20%) in wastewater (Schwartz et al. 2003; Volkmann et al. 2004; Shannon et al. 2007; Faria et al. 2009; Börjesson et al. 2010; Heß & Gallert 2014; Moges et al. 2014; Gómez et al. 2016). Only 2% of isolates were MRSA strains. Albeit the low occurrence of MRSA was reported in urban wastewater in Spain (Gómez et al. 2016) but most of the studies showed higher occurrence of MRSA strains, as in Iran (15.3%), Australia (50–55%), USA (68%), and Sweden (100%). Most of these studies have been on municipal and hospital wastewater (Börjesson et al. 2010; Goldstein et al. 2012; Thompson et al. 2013; Rahimi & Bouzari 2015).

Similar to some other reports around the world (Igimi et al. 1990; Abraham et al. 2007; Faria et al. 2009; Sasaki et al. 2010; Heß & Gallert 2014), S. intermedius (14.5%), S. hyicus (4.3%), and S. schleiferi subsp. coagulans (1.4%) were the other CoPS species identified in wastewater samples in our study. Most of them were isolated from livestock and poultry wastewater. This is in accordance with the fact that these species mainly colonize animal hosts and are responsible for infections such as pyoderma, otitis externa and genitourinary tract diseases in animals. However, they also cause opportunistic infections in humans (Sasaki et al. 2007, 2010).

Unless clinics, the antibiotic resistance in aquatic environments was less studied (Sharma et al. 2016). Extensive usage of antibiotics in hospitals, home therapy, veterinary and other areas hence continues the release of their residues into wastewater develops drug-resistant organisms (Heß & Gallert 2014). MDR Staphylococci (S. aureus and CoNS) has been detected in various environmental sources (Abulreesh 2011). In the present study, overall 50% of S. aureus isolates and 47% of CoNS isolates were MDR.

We found that S. aureus isolates were mostly penicillin resistance (89%). Similarly, penicillin resistance frequency was reported as 100% in S. aureus isolates collected from poultry and municipal wastewaters in Iran (Rahimi & Bouzari 2015; Rahimi & Karimi 2015). As well as the frequency of resistance against erythromycin, azithromycin and tetracycline were high in S. aureus and CoNS isolated from wastewaters. This finding is in agreement with other studies all over the world (Nawaz et al. 2000; Schwartz et al. 2003; Faria et al. 2009; Heß & Gallert 2014; Kumar et al. 2015).

Higher prevalence of macrolides, penicillin and tetracycline resistance among Staphylococci could be attributed to the expanded utilization of these agents in human and outside human medicine. In a systematic review performed in 2021, it was revealed that the median of antibiotic prescribing accounted for 45.25 and 68.2% of outpatient and inpatient settings in Iran, respectively. β-lactams (e.g., penicillins, cephalosporins, and carbapenems) and macrolides were the most commonly prescribed antibiotic classes (Nabovati et al. 2021). According to World Health Organization (WHO), about half of the worldwide-produced antibiotics are sold for use by outside humans (World Health Organization 2002). Medically important antibiotics were commonly used for the prophylactic purpose, growth promotion in food-producing animals and veterinary. In line with the global trends, tetracycline, penicillin and macrolides are among the most common antibiotics used in livestock and poultry farms in Iran (Alipour et al. 2014). Widespread usage of tetracycline, penicillin and macrolides both in humans and outside of human medicine could explain the high prevalence of resistance to these antibiotics in the current study.

Erythromycin-resistant Staphylococcus spp. isolates may display cross-resistance to clindamycin. About 11% of S. aureus isolates showed inducible clindamycin resistance in our study. Inducible resistance to clindamycin was reported as 19 and 32% in Germany and France, respectively (Lina et al. 1999; Heß & Gallert 2014). erm A, erm B, erm C, and erm TR genes are involved in erythromycin resistance in S. aureus by expressing methylase enzyme (Heß & Gallert 2014). While erm B gene is prominent in isolates from animal sources (Lina et al. 1999), erm C and erm A are dominant in clinical isolates (Fiebelkorn et al. 2003; Gherardi et al. 2009). Similarly, the frequency of erm C gene was high in our study (82.5%) and may be due to sources of our isolates mainly collected from hospital sewage. Also the erm B, erm TR, and msr A genes were found in our S. aureus isolates. Another mechanism to develop erythromycin resistance is to express msrA, a drug efflux pump-encoding gene (Heß & Gallert 2014). The msr A gene was found in nearly half of our isolates. It can be concluded that drug efflux along erm genes causes erythromycin resistance in current S. aureus isolates.

Resistance to aminoglycoside antibiotics tobramycin, kanamycin and gentamycin were identified in 11, 4 and 2% of S. aureus isolates, respectively. Aminoglycoside-resistant isolates carried aac (22.5%), ant (1%,) and aphc (10%) genes. Some of the previous studies reported that aac gene has the highest frequency among other genes encoding aminoglycoside resistance (Börjesson et al. 2010; Goldstein et al. 2012; Gómez et al. 2016).

Toxins play a vital role in the pathogenesis of staphylococcal infections. High variation in frequency of toxin encoding genes in S. aureus has been documented worldwide (Shallcross et al. 2013; Bhowmik et al. 2021). Wastewater pathogenic microbial population originates from human or animal excretions. Therefore, variations in the abundance of virulence genes in wastewater isolates in different regions may arise from the differences in virulence traits of the normal flora of the hosts. This can be approved by the fact that the pattern of virulence genes among our isolates is similar to the previous isolates collected in Ardabil from patients (unpublished data) and healthy people (Omid et al. 2021). It is clear that changing the sampling places can lead to differences in the virulence genes frequency.

In this study, the most prominent toxin genes were α-hemolysin encoding, hld (96%) and hla (94.5%) in S. aureus isolates. According to results reported by some previous studies, the hla and hld gene prevalence is 70–98% in Staphylococci isolates in clinical and non-clinical settings (Sharma-Kuinkel et al. 2015; Rossato 2018; Verdú-Expósito et al. 2020; Zieliński et al. 2020). α-hemolysin damages host cells by creating channels and disrupting the membrane (Tang et al. 2019). This toxin affects innate immune cells, disrupts epithelial and endothelial barriers, and stimulates a hyperinflammatory response following staphylococcal pneumonia (Tabor et al. 2016).

Several enterotoxins are responsible for food poisoning by S. aureus. Type A enterotoxin is the most common cause of food poisoning in humans (Argudín et al. 2010). In this study, sea, encoding type A staphylococcal enterotoxin was the predominant gene (87%) followed by sec (47%) encoding type C, sed (14.5%) encoding type D and seb (5.4%) encoding and B enterotoxins.

Toxic shock syndrome (TSS) is a deadly staphylococcal disease typically caused by the toxic shock syndrome toxin 1 (TSST-1) encoded by the tst gene (Parrish et al. 2019). We found tst gene in 84% of S. aureus isolates which is higher than other reports ranging 2–28.5% (Robert et al. 2011; Zieliński et al. 2020; Bhowmik et al. 2021).

The exfoliative toxins (encoded by eta and etb) cause the staphylococcal scaled-skin syndrome (SSSS). eta gene was carried by 33% of S. aureus isolates, while we could not detect etb gene. Previous studies have shown a higher prevalence (34–61%) of both of these genes in clinical and colonizing S. aureus isolates (Shukla et al. 2010; Champion et al. 2014).

Another studied gene was pvl encoding a two components toxin named Panton-Valentine leukocidin (PVL). PVL damages leukocytes membrane by creating pores. pvl was detected in 31% of isolates which locates in the range of earlier reported values (4.4–100%) (Shukla et al. 2010; Alfatemi et al. 2014; Papadimitriou-Olivgeris et al. 2017; Wang et al. 2017; Darboe et al. 2019; Veloso et al. 2019).

In the present study, eight isolates of S. aureus were assessed by spa typing approach in which six various types were identified including t346, t026, t14870, t937, t17068, and a new type t19215. Among them just t19215 belongs to MRSA strain. This finding is in agreement with our previous investigation on spa types in isolates from healthy people (Omid et al. 2021). Surveys on S. aureus isolates from wastewater in Spain and United States (Sauer et al. 2008; Friese et al. 2013) identified 15 spa types and just type t346 was identical to our result.

In the current study, multiple CoNS and CoPS species with veterinary and clinical significance were identified in the sewages from different sources. The most common CoPS and CoNS species were S. aureus and S. xylosus, respectively. A significant portion of both CoNS and CoPS isolates were resistant to multiple antibiotics. S. aureus isolates carried multiple virulence-encoding genes. The occurrence of these superbugs in wastewater is of great health concern indicating the importance of water treatment to eliminate antibiotic-resistant bacteria. We suggest using chemicals with high antimicrobial activity in wastewater processing in order to remove antibiotic-resistant bacteria and antibiotic resistance-encoding genes efficiently.

The authors wish to thank Mojtaba Amani, Professor of Biophysics from Ardabil University of Medical Sciences for his help in English-language editing of the manuscript.

The authors state that there was no specific funding for this work.

M.R.O. performed the experiments, contributed to the analysis of the results and drafted the manuscript. H.J. helped supervise the project and worked on the manuscript. F.K. helped in interpreting the results and worked on the manuscript. A.B. contributed to the final version of the manuscript. M.A. supervised the project, designed the study, conceived and planned the experiments, contributed to the analysis of the results, and took the lead in writing the manuscript.

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

The authors declare there is no conflict.