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Frequency of superantigen encoding genes of Staphylococcus aureus isolates collected from multiple sclerosis (MS) patients and nasal carriers
Microbial Pathogenesis 127 (2019) 316–319
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Frequency of superantigen encoding genes of Staphylococcus aureus isolates collected from multiple sclerosis (MS) patients and nasal carriers
T
Javid Sadeghia, Naser Alizadehb, Mahin Ahangar Oskoueia, Delara Laghusic, Daryush Savadi Oskoueid, Masoud Nikanfard, Mir Naser Seyyed Mousavie,f,∗ a
Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran c Department of Social Medicine, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Neurology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran e Department of Microbiology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran f Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Multiple sclerosis Staphylococcus aureus Super antigens
Background: Bacterial superantigens are potent T cell activators that can have acute or chronic effects on the central nervous system. Objectives: In this study, the role of enterotoxins, exfoliative toxins and toxic shock syndrome toxin of Staphylococcus aureus was investigated in MS patients and healthy nasal carriers. Methods: Three-hundred fifty nasal swabs were collected from healthy nasal carriers (n = 210) and MS (n = 140) patients. Staphylococcus aureus superantigens were detected by multiplex PCR. Antimicrobial susceptibility pattern was performed using disk diffusion method. Results: The highest rates of nasal colonization were seen in MS patients (46.42%). The rates of nasal colonization in the healthcare workers were 30.95%. The most commonly detected superantigens were SEA (31.5%), SEB (17.7%) and ETA (16.9%). The Staphylococcus aureus isolates had the highest levels of resistance against erythromycin (57.7%), clindamycin (55.4%) and co-trimoxazole (43.1%). All isolates were susceptible to vancomycin, linezolid, and mupirocin. Conclusion: Our results revealed that the frequency of superantigen producing Staphylococcus aureus isolates is high in the MS patients. As well as these isolates are sensitive to mupirocin. Thus it is better to use of mupirocin for nasal decolonization of Staphylococcus aureus in the MS patients.
1. Introduction
superantigens effectively activate CD4+ T cells, containing massive cell proliferation and cytokine production, mainly IL-2 and interferon (IFN)γ [6–8]. The effects of these superantigens on the immune system can be both acute and long-term disease, and its effects contain deregulation of the immune response resulting in the proliferation of autoreactive T cells and the development and/or exacerbation of chronic autoimmune diseases such as Multiple sclerosis (MS) [9,10]. MS is an autoimmune inflammatory demyelinating disease of the central nervous system (CNS) [11]. Although the precise etiology of MS is not known, the role of genetic and environmental factors in the development of MS has been shown [12]. Genetic factors for MS contain the human leukocyte antigen (HLA) loci and non-HLA genomic regions. From environmental factors, the role of infectious agents such as
Staphylococcus aureus (S. aureus) is a main human pathogenic bacteria capable of causing a wide range of infections, such as skin and ulcer infections; deep-seated and systemic infections [1]. The anterior nares are common sites of colonization by S. aureus and approximately 10%–40% of the people carries this bacterium [2]. Colonization of this organism is an important risk factor for infections [3]. Nasal colonized S. aureus strains can produce several virulence factors including superantigens (SAgs) such as staphylococcal enterotoxins (SEs), toxic shock syndrome toxin-1 (TSST-1), and exfoliative toxins (ETs) which are involved in different pathologies [4,5]. These superantigens are the most potent T cell mitogens ever discovered. Staphylococcal ∗
Corresponding author. Department of Microbiology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran. E-mail addresses: [email protected] (J. Sadeghi), [email protected] (N. Alizadeh), [email protected] (M. Ahangar Oskouei), [email protected] (D. Laghusi), [email protected] (D. Savadi Oskouei), [email protected] (M. Nikanfar), [email protected] (M.N. Seyyed Mousavi). https://doi.org/10.1016/j.micpath.2018.12.010 Received 26 April 2018; Received in revised form 5 December 2018; Accepted 6 December 2018 Available online 13 December 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 127 (2019) 316–319
J. Sadeghi et al.
bacteria, bacterial superantigens, viruses, and protozoa has been described [12]. Among bacterial superantigens, the relevance of S. aureus superantigens with MS exacerbation has been shown [5]. The correlation between bacterial superantigens and MS was initially based on experiments using the experimental autoimmune encephalomyelitis (EAE) model. Despite experimental studies, several studies have shown the association of nasal colonization with superantigen-producing S. aureus and MS exacerbation [5,13,14]. On the bases of our searching, in Iran, only one study has been published in this context [15]. The aim of this study was to investigate the prevalence of S. aureus colonization, the frequency of superantigens, and antibiotic susceptibility profile of these isolates between the MS patients and healthy nasal carriers of healthcare workers.
Table 1 Patient characteristics, overall and by MS patients and nasal carriers. Patient characteristics
S. aureus positive Gender Male Female Average Age MS family history
Study groups MS patients
Nasal carriers
Pvalue
140 (46/42%)
210 (30/95%)
0.003
15 (23/1%) 50 (76/9%) 34/12 4 (6/2%)
37 (56/9%) 28 (43/1%) 30/9 –
0.001 0.001 0.045
15, SPSS, Chicago, USA). Chi-square exact test and Fisher's exact twotailed test were used to test for significant associations between MS patients and healthy nasal carriers. p values less than 0.05 were considered significant.
2. Materials and methods 2.1. Collection of specimens
3. Results
From October 2016 to March 2017, nasal swabs were collected from MS patients (n = 140) who did not have a hospital stay of more than 2 days and healthcare workers (n = 210). All samples were collected from Emam Reza and Razi educational hospitals of Tabriz, Iran. The swab samples were rapidly transferred to the laboratory. This study was approved by The Ethics Commission of Tabriz University of Medical Sciences (5/42038/)ﺩ.
The study enrolled 130 isolates, 65 in the non-MS (healthy nasal carrier), 65 in the MS patients groups. Of 140 nasal swabs collected for MS patients, 65 (46.42%) were positive for S. aureus as well as 210 nasal swabs collected from healthy nasal carriers that 65 (30.95%) were positive. From 65 MS patients were positive for S. aureus, 59 patients had received immunosuppressing drugs including Cinnovex 26.2%, Rebif 18.5%, Fingolimode and Corticosteroid 13.8%, Gabapentin 7.7%, Betaferron 7.7% and Azathioprine 3.1%. Six patients (9.2%) were recently diagnosed and they did not receive drugs. On the other hand, the most commonly colonized group was MS patients group. Demographic data of the patients with MS and nasal carrier study groups are shown in Table 1. Distribution of super antigen genes of the S. aureus isolates of study subjects is shown in Table 2. The most commonly detected enterotoxins were sea (40%), seb (27.7%), and eta (11.11%). Significant differences were seen between the prevalence of sea (P = 0.038) and seb (P = 0.003) between MS patients and healthy nasal carriers. Antibiotic susceptibility pattern of S. aureus isolated from various studied groups is shown in Table 3. We found that the S. aureus strains of MS group had the highest levels of resistance to erythromycin (63.1%), clindamycin (60%) and co-trimoxazole (43.1%). The most effective antibiotics were vancomycin, mupirocin and linezolid.
2.2. Bacterial isolates For the isolation of S. aureus, swabs were inoculated onto 5% sheep blood agar (Liofilchem, ltd., Italy) and mannitol salt agar (Hi Media Co., India) plates and incubated at 35 °C for 48 h. These isolates were identified by Gram staining, catalase, coagulase, DNase activities, and mannitol salt agar tests. 2.3. Antimicrobial susceptibility testing Antimicrobial susceptibility test was performed by disk diffusion method according to the clinical and laboratory standards institute (CLSI) guidelines [16], with a panel of following antibiotics: gentamicin (10 μg), erythromycin (15 μg), cefoxitin (30 μg), Cefazolin (30 μg), ciprofloxacin (5 μg), Clindamycin (2 μg), mupirocin (200 μg), linezolid (5 μg), and trimethoprim/sulfamethoxazole (25 μg). All antibiotic disks were provided from Mast, Group Ltd, Merseyside, UK. MIC of vancomycin was determined by agar dilution method.
4. Discussion The role of Staphylococcal superantigens in several autoimmune diseases such as MS and Wegners has been investigated [5,13,19,20]. Nasal colonization with superantigen producing S. aureus even in the absence of infection can be triggered CD4+ cell response, which may have a major role in the exacerbation of MS disease [13]. In this study, the colonization of nasal with S. aureus in MS patients was more than healthy nasal carriers. This is may be due to more contact with these patients with healthcare settings and consumption of immunosuppressive drugs. Our results were in agreement with the results of other studies [13,15]. Superantigens directly interact with the variable region of the beta chain (Vβ) of the T cell receptor (TCR). To date, more than 60 different Vβ fragments of human TCRs have been recognized. Superantigens have differed in the activation of human T cells expressing diverse Vβ fragments [5,14]. For instance, staphylococcal toxic shock syndrome toxin-1 (TSST-1) specifically interacts with human Vβ2 T cells, whereas SEB and SEC3 are more promiscuous in their Vβ-targets and activates T cells bearing Vβ3, Vβ12, Vβ14, Vβ15, Vβ17, and Vβ20 [5,12,14]. Hence superantigens activate more than 20% of cells in a specified T cell population and they could have a potential role on the exacerbation or attenuation of MS [5,21]. Several studies have been shown the influence of staphylococcal superantigens in the MS exacerbation and
2.4. DNA extraction DNA extraction was performed by the method as previously described [17]. 2.5. Multiplex PCR for detection of super antigen genes Multiplex PCR was carried out for detection of superantigen genes of S. aureus include sea, seb, sec, sed, tst, eta, etb according to previously described method [18]. Specific primers and master mix were purchased from CinnaClone, Tehran, Iran. Multiplex PCR amplification of superantigen genes was performed in the Thermal Cycler (Bio RAD, T100™) under following conditions: 95 °C 15 min, 32 cycles with 95 °C 60 s, 60 °C 90 s, 72 °C 60 s, and finally, 72 °C 10 min. Multiplex PCR products were detected by agarose gel electrophoresis (1.2%). Previously detected clinical strains of S. aureus harboring studying genes were used as positive controls for PCR amplification. 2.6. Statistical analysis Data analysis was performed by SPSS statistical software (Version 317
Microbial Pathogenesis 127 (2019) 316–319
J. Sadeghi et al.
Table 2 Frequency of superantigens genes of S. aureus in studied groups. Study groups
MS patients Nasal carriers Total Pvalue
Genes sea
seb
sec
sed
tst
eta
etb
26 (40%) 15 (23/1%) 41 (31/5%) 0.038
18 (27/7%) 5 (7/7%) 23 (17/7%) 0.003
10 (15/4%) 4 (6/2%) 14 (10/8%) 0.090
3 (4/6%) 1 (1/5%) 4 (3/1%) 0.242
5 (7/7%) 12 (18/5%) 17 (13/1%) 0.069
14 (21/5%) 8 (12/3%) 22 (16/9%) 0.160
9 (13/8%) 5 (7/7%) 14 (10/8%) 0.258
Table 3 Antibiotic susceptibility patterna of S. aureus isolates in MS patients and nasal carriers. Study groups
MS patients Nasal carriers Total Pvalue
Antibiotics E
CD
SXT
CZ
CIP
G
FOX
41 (63/1%) 34 (52/3%) 75 (57/7%) 0.214
39 (60%) 33 (50/8%) 72 (55/4%) 0.290
28 (43/1%) 38 (58/5%) 66 (50/8%) 0.079
22 (33/8%) 14 (21/5%) 36 (27/7%) 0.117
16 (24/6%) 11 (16/9%) 27 (20/8%) 0.280
16 (24/6%) 10 (15/4%) 26 (20%) 0.188
0 (0%) 2 (3/1%) 2 (1/5%) 0.496
E: Erythromycin, CD: Clindamycin, SXT: Trimethoprim/sulfamethoxazole, CZ: Cefazolin, CIP: Ciprofloxacin, G: Gentamicin, FOX: Cefoxitin. a All isolates were susceptible to vancomycin, linzolid and mupirocin.
doi.org/10.1016/j.micpath.2018.12.010.
attenuation [13,15,22]. In this study, seven superantigenic genes (sea, seb, sec, sed, tst, eta, and etb) were identified in the S. aureus isolates. The frequency of S. aureus harboring sea, seb, sec, sed, eta, and etb genes in MS patients was higher than that of healthy nasal carriers. But the frequency of S. aureus harboring tst gene in MS patients was lower than that of healthy nasal carriers. The significant association was observed in S. aureus harboring sea (p < 0.038) and seb (p < 0.003) genes between MS and healthy nasal carriers. There was no significant association among other studied genes in S. aureus isolates between MS and healthy nasal carriers. The results of this study were in accordance with the results of Mulvey and Mehrabi studies [13,15]. Mupirocin is the most antimicrobial agent that has been applied to the nasal decolonization of S. aureus in individuals that have been a risk factor for S. aureus disease [23]. In this study, all S. aureus isolates were susceptible to vancomycin, linzolid and mupirocin. Therefore, mupirocin could be applied to the nasal decolonization of S. aureus in MS patients. Nasal colonization with methicillin resistant S. aureus (MRSA) can increase the risk of MRSA infection. Approximately 1% of total population is colonized with MRSA strains [23]. In the present study, 3.1% of healthy nasal carriers harbored MRSA strains, whereas no MRSA strain was detected in MS patients. The results of this study showed that the frequency of S. aureus superantigens in MS patients is more than that of healthy nasal carriers. Hence it shows the probable role of staphylococcal superantigens in MS exacerbation. Also, the presence of staphylococcal superantigens in healthy nasal carriers of hospital personnel can be a risk factor for MS patients. It is better to have MS patients away from polluted hospital and health care center environments. It is also recommended to use mupirocin ointment in the nose to prevent the exacerbation of MS.
References [1] M.T. Akhi, R. Ghotaslou, M.Y. Memar, M. Asgharzadeh, M. Varshochi, T. Pirzadeh, et al., Frequency of MRSA in diabetic foot infections, Int. J. Diabetes Dev. Ctries. 37 (1) (2017) 58–62. [2] J. Kluytmans, A. Van Belkum, H. Verbrugh, Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks, Clin. Microbiol. Rev. 10 (3) (1997) 505–520. [3] S. Holtfreter, D. Grumann, M. Schmudde, H. Nguyen, P. Eichler, B. Strommenger, et al., Clonal distribution of superantigen genes in clinical Staphylococcus aureus isolates, J. Clin. Microbiol. 45 (8) (2007) 2669–2680. [4] M.M. Dings, P.M. Orwin, P. Schlievert, Exotoxins of Staphyloccocus aureus, Clin. Microbiol. Rev. 13 (2000) 16–34. [5] B.A. Torres, S. Kominsky, G.Q. Perrin, A.C. Hobeika, H.M. Johnson, Superantigens: the good, the bad, and the ugly, Exp. Biol. Med. 226 (3) (2001) 164–176. [6] T. Proft, J.D. Fraser, Bacterial superantigens, Clin. Exp. Immunol. 133 (3) (2003) 299–306. [7] K. Ito, H. Takaishi, Y. Jin, F. Song, T.L. Denning, P.B. Ernst, Staphylococcal enterotoxin B stimulates expansion of autoreactive T cells that induce apoptosis in intestinal epithelial cells: regulation of autoreactive responses by IL-10, J. Immunol. 164 (6) (2000) 2994–3001. [8] H. Li, A. Llera, D. Tsuchiya, L. Leder, X. Ysern, P.M. Schlievert, et al., Three-dimensional structure of the complex between a T cell receptor β chain and the superantigen staphylococcal enterotoxin B, Immunity 9 (6) (1998) 807–816. [9] P.M. Schlievert, Role of superantigens in human disease, J. Infect. Dis. 167 (5) (1993) 997–1002. [10] T.J. Foster, Immune evasion by staphylococci, Nat. Rev. Microbiol. 3 (12) (2005) 948–958. [11] J. Fletcher, S. Lalor, C. Sweeney, N. Tubridy, K. Mills, T cells in multiple sclerosis and experimental autoimmune encephalomyelitis, Clin. Exp. Immunol. 162 (1) (2010) 1–11. [12] J.E. Libbey, M.F. Cusick, R.S. Fujinami, Role of pathogens in multiple sclerosis, Int. Rev. Immunol. 33 (4) (2014) 266–283. [13] M.R. Mulvey, M. Doupe, M. Prout, C. Leong, R. Hizon, A. Grossberndt, et al., Staphylococcus aureus harbouring Enterotoxin A as a possible risk factor for multiple sclerosis exacerbations, Mult. Scler. J 17 (4) (2011) 397–403. [14] S.X. Xu, J.K. McCormick, Staphylococcal superantigens in colonization and disease, Front Cell Inf. Microb. 2 (2012) 52, https://doi.org/10.3389/fcimb. 2012.00052. [15] F. Mehrabi, A. Asgari, Resistant Strains of Enterotoxigenic Staphylococcus aureus; Unknown Risk for Multiple Sclerosis Exacerbation, Iran. Red Crescent Med. J. 17 (9) (2015) e12596. [16] P. Wayne, Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing, Clinical and Laboratory Standards Institute, 2015, p. 21. [17] J. Sadeghi, S. Mansouri, Molecular characterization and antibiotic resistance of clinical isolates of methicillin‐resistant Staphylococcus aureus obtained from Southeast of Iran (Kerman), APMIS 122 (5) (2014) 405–411. [18] G. Echaniz‐Aviles, M. Velazquez‐Meza, M. Aires‐de‐Sousa, R. Morfín‐Otero, E. Rodríguez‐Noriega, N. Carnalla‐Barajas, et al., Molecular characterisation of a dominant methicillin‐resistant Staphylococcus aureus (MRSA) clone in a Mexican hospital (1999–2003), Clin. Microbiol. Infect. 12 (1) (2006) 22–28. [19] J. Li, J. Yang, Y-w Lu, S. Wu, M-r Wang, J-m Zhu, Possible role of staphylococcal
Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgements This article was financially supported by the Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// 318
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[22] T.G.D. França, F. Chiuso-Minicucci, S.F.G. Zorzella-Pezavento, L.L.W. Ishikawa, L.C. Rosa, P.M. Colavite, et al., Previous infection with Staphylococcus aureus strains attenuated experimental encephalomyelitis, BMC Neurosci. 15 (2014) 8. [23] T. Coates, R. Bax, A. Coates, Nasal decolonization of Staphylococcus aureus with mupirocin: strengths, weaknesses and future prospects, J. Antimicrob. Chemother. 64 (2009) 9–15.
enterotoxin B in the pathogenesis of autoimmune diseases, Viral Immunol. 28 (7) (2015) 354–359. [20] M.N.S. Mousavi, B. Mehramuz, J. Sadeghi, N. Alizadeh, M.A. Oskouee, H.S. Kafil, The pathogenesis of Staphylococcus aureus in autoimmune diseases, Microb. Pathog. 111 (2017). [21] P. Marrack, J. Kappler, The staphylococcal enterotoxins and their relatives, Science 248 (1990) 705–711.
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Frequency of superantigen encoding genes of Staphylococcus aureus isolates collected from multiple sclerosis (MS) patients and nasal carriers
T
Javid Sadeghia, Naser Alizadehb, Mahin Ahangar Oskoueia, Delara Laghusic, Daryush Savadi Oskoueid, Masoud Nikanfard, Mir Naser Seyyed Mousavie,f,∗ a
Department of Bacteriology and Virology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran c Department of Social Medicine, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Neurology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran e Department of Microbiology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran f Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Multiple sclerosis Staphylococcus aureus Super antigens
Background: Bacterial superantigens are potent T cell activators that can have acute or chronic effects on the central nervous system. Objectives: In this study, the role of enterotoxins, exfoliative toxins and toxic shock syndrome toxin of Staphylococcus aureus was investigated in MS patients and healthy nasal carriers. Methods: Three-hundred fifty nasal swabs were collected from healthy nasal carriers (n = 210) and MS (n = 140) patients. Staphylococcus aureus superantigens were detected by multiplex PCR. Antimicrobial susceptibility pattern was performed using disk diffusion method. Results: The highest rates of nasal colonization were seen in MS patients (46.42%). The rates of nasal colonization in the healthcare workers were 30.95%. The most commonly detected superantigens were SEA (31.5%), SEB (17.7%) and ETA (16.9%). The Staphylococcus aureus isolates had the highest levels of resistance against erythromycin (57.7%), clindamycin (55.4%) and co-trimoxazole (43.1%). All isolates were susceptible to vancomycin, linezolid, and mupirocin. Conclusion: Our results revealed that the frequency of superantigen producing Staphylococcus aureus isolates is high in the MS patients. As well as these isolates are sensitive to mupirocin. Thus it is better to use of mupirocin for nasal decolonization of Staphylococcus aureus in the MS patients.
1. Introduction
superantigens effectively activate CD4+ T cells, containing massive cell proliferation and cytokine production, mainly IL-2 and interferon (IFN)γ [6–8]. The effects of these superantigens on the immune system can be both acute and long-term disease, and its effects contain deregulation of the immune response resulting in the proliferation of autoreactive T cells and the development and/or exacerbation of chronic autoimmune diseases such as Multiple sclerosis (MS) [9,10]. MS is an autoimmune inflammatory demyelinating disease of the central nervous system (CNS) [11]. Although the precise etiology of MS is not known, the role of genetic and environmental factors in the development of MS has been shown [12]. Genetic factors for MS contain the human leukocyte antigen (HLA) loci and non-HLA genomic regions. From environmental factors, the role of infectious agents such as
Staphylococcus aureus (S. aureus) is a main human pathogenic bacteria capable of causing a wide range of infections, such as skin and ulcer infections; deep-seated and systemic infections [1]. The anterior nares are common sites of colonization by S. aureus and approximately 10%–40% of the people carries this bacterium [2]. Colonization of this organism is an important risk factor for infections [3]. Nasal colonized S. aureus strains can produce several virulence factors including superantigens (SAgs) such as staphylococcal enterotoxins (SEs), toxic shock syndrome toxin-1 (TSST-1), and exfoliative toxins (ETs) which are involved in different pathologies [4,5]. These superantigens are the most potent T cell mitogens ever discovered. Staphylococcal ∗
Corresponding author. Department of Microbiology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran. E-mail addresses: [email protected] (J. Sadeghi), [email protected] (N. Alizadeh), [email protected] (M. Ahangar Oskouei), [email protected] (D. Laghusi), [email protected] (D. Savadi Oskouei), [email protected] (M. Nikanfar), [email protected] (M.N. Seyyed Mousavi). https://doi.org/10.1016/j.micpath.2018.12.010 Received 26 April 2018; Received in revised form 5 December 2018; Accepted 6 December 2018 Available online 13 December 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 127 (2019) 316–319
J. Sadeghi et al.
bacteria, bacterial superantigens, viruses, and protozoa has been described [12]. Among bacterial superantigens, the relevance of S. aureus superantigens with MS exacerbation has been shown [5]. The correlation between bacterial superantigens and MS was initially based on experiments using the experimental autoimmune encephalomyelitis (EAE) model. Despite experimental studies, several studies have shown the association of nasal colonization with superantigen-producing S. aureus and MS exacerbation [5,13,14]. On the bases of our searching, in Iran, only one study has been published in this context [15]. The aim of this study was to investigate the prevalence of S. aureus colonization, the frequency of superantigens, and antibiotic susceptibility profile of these isolates between the MS patients and healthy nasal carriers of healthcare workers.
Table 1 Patient characteristics, overall and by MS patients and nasal carriers. Patient characteristics
S. aureus positive Gender Male Female Average Age MS family history
Study groups MS patients
Nasal carriers
Pvalue
140 (46/42%)
210 (30/95%)
0.003
15 (23/1%) 50 (76/9%) 34/12 4 (6/2%)
37 (56/9%) 28 (43/1%) 30/9 –
0.001 0.001 0.045
15, SPSS, Chicago, USA). Chi-square exact test and Fisher's exact twotailed test were used to test for significant associations between MS patients and healthy nasal carriers. p values less than 0.05 were considered significant.
2. Materials and methods 2.1. Collection of specimens
3. Results
From October 2016 to March 2017, nasal swabs were collected from MS patients (n = 140) who did not have a hospital stay of more than 2 days and healthcare workers (n = 210). All samples were collected from Emam Reza and Razi educational hospitals of Tabriz, Iran. The swab samples were rapidly transferred to the laboratory. This study was approved by The Ethics Commission of Tabriz University of Medical Sciences (5/42038/)ﺩ.
The study enrolled 130 isolates, 65 in the non-MS (healthy nasal carrier), 65 in the MS patients groups. Of 140 nasal swabs collected for MS patients, 65 (46.42%) were positive for S. aureus as well as 210 nasal swabs collected from healthy nasal carriers that 65 (30.95%) were positive. From 65 MS patients were positive for S. aureus, 59 patients had received immunosuppressing drugs including Cinnovex 26.2%, Rebif 18.5%, Fingolimode and Corticosteroid 13.8%, Gabapentin 7.7%, Betaferron 7.7% and Azathioprine 3.1%. Six patients (9.2%) were recently diagnosed and they did not receive drugs. On the other hand, the most commonly colonized group was MS patients group. Demographic data of the patients with MS and nasal carrier study groups are shown in Table 1. Distribution of super antigen genes of the S. aureus isolates of study subjects is shown in Table 2. The most commonly detected enterotoxins were sea (40%), seb (27.7%), and eta (11.11%). Significant differences were seen between the prevalence of sea (P = 0.038) and seb (P = 0.003) between MS patients and healthy nasal carriers. Antibiotic susceptibility pattern of S. aureus isolated from various studied groups is shown in Table 3. We found that the S. aureus strains of MS group had the highest levels of resistance to erythromycin (63.1%), clindamycin (60%) and co-trimoxazole (43.1%). The most effective antibiotics were vancomycin, mupirocin and linezolid.
2.2. Bacterial isolates For the isolation of S. aureus, swabs were inoculated onto 5% sheep blood agar (Liofilchem, ltd., Italy) and mannitol salt agar (Hi Media Co., India) plates and incubated at 35 °C for 48 h. These isolates were identified by Gram staining, catalase, coagulase, DNase activities, and mannitol salt agar tests. 2.3. Antimicrobial susceptibility testing Antimicrobial susceptibility test was performed by disk diffusion method according to the clinical and laboratory standards institute (CLSI) guidelines [16], with a panel of following antibiotics: gentamicin (10 μg), erythromycin (15 μg), cefoxitin (30 μg), Cefazolin (30 μg), ciprofloxacin (5 μg), Clindamycin (2 μg), mupirocin (200 μg), linezolid (5 μg), and trimethoprim/sulfamethoxazole (25 μg). All antibiotic disks were provided from Mast, Group Ltd, Merseyside, UK. MIC of vancomycin was determined by agar dilution method.
4. Discussion The role of Staphylococcal superantigens in several autoimmune diseases such as MS and Wegners has been investigated [5,13,19,20]. Nasal colonization with superantigen producing S. aureus even in the absence of infection can be triggered CD4+ cell response, which may have a major role in the exacerbation of MS disease [13]. In this study, the colonization of nasal with S. aureus in MS patients was more than healthy nasal carriers. This is may be due to more contact with these patients with healthcare settings and consumption of immunosuppressive drugs. Our results were in agreement with the results of other studies [13,15]. Superantigens directly interact with the variable region of the beta chain (Vβ) of the T cell receptor (TCR). To date, more than 60 different Vβ fragments of human TCRs have been recognized. Superantigens have differed in the activation of human T cells expressing diverse Vβ fragments [5,14]. For instance, staphylococcal toxic shock syndrome toxin-1 (TSST-1) specifically interacts with human Vβ2 T cells, whereas SEB and SEC3 are more promiscuous in their Vβ-targets and activates T cells bearing Vβ3, Vβ12, Vβ14, Vβ15, Vβ17, and Vβ20 [5,12,14]. Hence superantigens activate more than 20% of cells in a specified T cell population and they could have a potential role on the exacerbation or attenuation of MS [5,21]. Several studies have been shown the influence of staphylococcal superantigens in the MS exacerbation and
2.4. DNA extraction DNA extraction was performed by the method as previously described [17]. 2.5. Multiplex PCR for detection of super antigen genes Multiplex PCR was carried out for detection of superantigen genes of S. aureus include sea, seb, sec, sed, tst, eta, etb according to previously described method [18]. Specific primers and master mix were purchased from CinnaClone, Tehran, Iran. Multiplex PCR amplification of superantigen genes was performed in the Thermal Cycler (Bio RAD, T100™) under following conditions: 95 °C 15 min, 32 cycles with 95 °C 60 s, 60 °C 90 s, 72 °C 60 s, and finally, 72 °C 10 min. Multiplex PCR products were detected by agarose gel electrophoresis (1.2%). Previously detected clinical strains of S. aureus harboring studying genes were used as positive controls for PCR amplification. 2.6. Statistical analysis Data analysis was performed by SPSS statistical software (Version 317
Microbial Pathogenesis 127 (2019) 316–319
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Table 2 Frequency of superantigens genes of S. aureus in studied groups. Study groups
MS patients Nasal carriers Total Pvalue
Genes sea
seb
sec
sed
tst
eta
etb
26 (40%) 15 (23/1%) 41 (31/5%) 0.038
18 (27/7%) 5 (7/7%) 23 (17/7%) 0.003
10 (15/4%) 4 (6/2%) 14 (10/8%) 0.090
3 (4/6%) 1 (1/5%) 4 (3/1%) 0.242
5 (7/7%) 12 (18/5%) 17 (13/1%) 0.069
14 (21/5%) 8 (12/3%) 22 (16/9%) 0.160
9 (13/8%) 5 (7/7%) 14 (10/8%) 0.258
Table 3 Antibiotic susceptibility patterna of S. aureus isolates in MS patients and nasal carriers. Study groups
MS patients Nasal carriers Total Pvalue
Antibiotics E
CD
SXT
CZ
CIP
G
FOX
41 (63/1%) 34 (52/3%) 75 (57/7%) 0.214
39 (60%) 33 (50/8%) 72 (55/4%) 0.290
28 (43/1%) 38 (58/5%) 66 (50/8%) 0.079
22 (33/8%) 14 (21/5%) 36 (27/7%) 0.117
16 (24/6%) 11 (16/9%) 27 (20/8%) 0.280
16 (24/6%) 10 (15/4%) 26 (20%) 0.188
0 (0%) 2 (3/1%) 2 (1/5%) 0.496
E: Erythromycin, CD: Clindamycin, SXT: Trimethoprim/sulfamethoxazole, CZ: Cefazolin, CIP: Ciprofloxacin, G: Gentamicin, FOX: Cefoxitin. a All isolates were susceptible to vancomycin, linzolid and mupirocin.
doi.org/10.1016/j.micpath.2018.12.010.
attenuation [13,15,22]. In this study, seven superantigenic genes (sea, seb, sec, sed, tst, eta, and etb) were identified in the S. aureus isolates. The frequency of S. aureus harboring sea, seb, sec, sed, eta, and etb genes in MS patients was higher than that of healthy nasal carriers. But the frequency of S. aureus harboring tst gene in MS patients was lower than that of healthy nasal carriers. The significant association was observed in S. aureus harboring sea (p < 0.038) and seb (p < 0.003) genes between MS and healthy nasal carriers. There was no significant association among other studied genes in S. aureus isolates between MS and healthy nasal carriers. The results of this study were in accordance with the results of Mulvey and Mehrabi studies [13,15]. Mupirocin is the most antimicrobial agent that has been applied to the nasal decolonization of S. aureus in individuals that have been a risk factor for S. aureus disease [23]. In this study, all S. aureus isolates were susceptible to vancomycin, linzolid and mupirocin. Therefore, mupirocin could be applied to the nasal decolonization of S. aureus in MS patients. Nasal colonization with methicillin resistant S. aureus (MRSA) can increase the risk of MRSA infection. Approximately 1% of total population is colonized with MRSA strains [23]. In the present study, 3.1% of healthy nasal carriers harbored MRSA strains, whereas no MRSA strain was detected in MS patients. The results of this study showed that the frequency of S. aureus superantigens in MS patients is more than that of healthy nasal carriers. Hence it shows the probable role of staphylococcal superantigens in MS exacerbation. Also, the presence of staphylococcal superantigens in healthy nasal carriers of hospital personnel can be a risk factor for MS patients. It is better to have MS patients away from polluted hospital and health care center environments. It is also recommended to use mupirocin ointment in the nose to prevent the exacerbation of MS.
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