Coexistence of virulence genes in methicillin-resistant Staphylococcus aureus clinical isolates

Adriana Medianeira Rossato Keli Cristine Reiter Pedro Alves d’Azevedo About the authors

Abstract

INTRODUCTION:

The pathogenic versatility of Staphylococcus aureus is attributed to various virulence genes, including enterotoxins and hemolysins.

METHODS:

Here, the virulence genes in 177 nosocomial MRSA strains in Porto Alegre, Brazil were detected by PCR.

RESULTS:

The overall prevalence rates were as follows: sea, 4.5%; pvl, 18.6%; tst, 27.7%; hla, 87.6%; and hld, 90.4%. No strain contained all tested genes. However, there was frequent coexistence of tst with pvl and hla with hld (40.7% and 26.6%, respectively).

CONCLUSIONS:

Horizontal transfer of virulence genes is very common in S. aureus, as suggested by the frequent coexistence of several virulence genes.

Keywords:
Coexistence of virulence genes; MRSA; Virulence genes

Virulence and antimicrobial resistance are two characteristics that do not always coexist in bacteria, since both are associated with large metabolic burdens, resulting in decreased fitness11. Händel N, Schuurmans JM, Brul S, ter Kuile BH. Compensation of the metabolic costs of antibiotic resistance by physiological adaptation in Escherichia coli. Antimicrob Agents Chemother. 2013;57(8):3752-62.. However, if the microorganism harbors the genetic capacity allowing it to produce either the determinants of antimicrobial resistance or virulence factors in specific situations, if a host demonstrate weakness, then both might be concomitantly expressed. Staphylococcus aureus has several resistance mechanisms and several virulence genes that promote adhesion to host cells, tissue invasion, and escape from the immune system, to establish a chronic infection22. Tong SYC, Chen LF, Fowler VG. Colonization, pathogenicity, host susceptibility, and therapeutics for Staphylococcus aureus: what is the clinical relevance? Seminars in Immunopathology. 2012;34(2):185-200..

Methicillin resistance is the most prevalent resistance acquired by S. aureus, and it is highly disseminated worldwide. As an opportunistic microorganism, S. aureus takes advantage of its existence as both a commensal and a pathogen to survive in the host, since selective pressures promote adaptation. Moreover, when living with other commensal bacteria, such as Staphylococcus epidermidis and Enterococcus, S. aureus may acquire additional resistance or virulence characteristics11. Händel N, Schuurmans JM, Brul S, ter Kuile BH. Compensation of the metabolic costs of antibiotic resistance by physiological adaptation in Escherichia coli. Antimicrob Agents Chemother. 2013;57(8):3752-62.,33. Sollid JU, Furberg AS, Hanssen AM, Johannessen M. Staphylococcus aureus: determinants of human carriage. Infect Genet Evol. 2014;21:531-41.. Because many of these genes are encoded on mobile genetic elements, such as plasmids or prophages, they can be transmitted between strains by horizontal transfer44. Otto M. Staphylococcus aureus toxins. Curr Opin Microbiol. 2014;17:32-7.. In addition to its antimicrobial resistance arsenal, there are several virulence genes that make S. aureus such a versatile pathogen. The most frequent virulence genes encode toxins such as enterotoxins, Panton-Valentine leukocidin, and toxic shock syndrome toxin, and hemolysins such as alpha-, beta-, and delta-hemolysins. The expression of these virulence factors is coordinated by quorum-sensing activity, a cell-communication system that controls gene expression in response to population density55. Yu F, Liu Y, Lv J, Qi X, Lu C, Ding Y. Antimicrobial susceptibility, virulence determinant carriage and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections. Braz J Infect Dis. 2015;19(6):614-22.,66. Kong C, Neoh HM, Nathan S. Targeting Staphylococcus aureus toxins: a potential form of anti-virulence therapy. Toxins (Basel). 2016;8(3):72..

In this study, we report the coexistence of quorum-sensing regulated virulence genes among a collection of methicillin-resistant Staphylococcus aureus (MRSA) strains obtained from hospitals in Porto Alegre, Brazil. Our findings provide insight into the pathogenesis and evolution of MRSA.

This cross-sectional observational study was conducted with 177 healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) strains recovered between 2012 and 2014 in Laboratories from Hospital Mãe de Deus (HMD) and Hospital Nossa Senhora da Conceição (HNSC). They were isolated from the respiratory tract (72/177; 40.7%), blood (40/177; 22.6%), skin and soft tissue (39/177; 22.0%), bone and connective tissue (10/177; 5.6%), medical devices (9/177; 5.1%), urine (5/177; 2.8%), and others sites (2/177; 1.1%). The identity of the isolates was confirmed using conventional methods, such as Gram staining, catalase activity, and plasma coagulase production. Methicillin resistance was verified by conventional polymerase chain reaction (PCR) for mecA and by the Kirby-Bauer method using cefoxitin (DME, São Paulo, Brazil), according to Clinical and Laboratory Standard Institute (CLSI) guidelines77. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: twenty-fourth informational supplement. Document M100-S24. Wayne, PA: CLSI; 2015. p.124-39..

Deoxyribonucleic acid (DNA) was extracted from MRSA isolates grown for 24h using Chelex® 100 (Bio-Rad, Richmond, CA) and Proteinase K (Sigma-Aldrich, Poole, UK) and was subjected to qualitative PCR. The alpha-hemolysin (hla); delta-hemolysin (hld); staphylococcal enterotoxin type A (sea); Panton-Valentine leucocidin (pvl), and toxic shock syndrome toxin-1 (tst) virulence genes were detected according to a previously described method88. Hoseini Alfatemi SM, Motamedifar M, Hadi N, Sedigh Ebrahim Saraie H. Analysis of virulence genes among methicillin-resistant Staphylococcus aureus (MRSA) Strains. Jundishapur J Microbiol. 2014;7(6):e10741., with some modifications. The PCR mixture contained 2.75mM MgCl2, 0.2mM each deoxyribonucleotide triphosphate, 1× Taq buffer, 1.25U of Taq DNA polymerase, 0.5μM primers, and 1.8μl of DNA template in a total volume of 15μl. The thermal cycler program used for sea, hla, hld, and tst was as follows: an initial denaturation step at 94°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 50°C (tst), 52°C (sea), or 58°C (hla, hld) for 45 sec, and extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The thermal cycler program used for pvl was as follows: an initial denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 15 sec, annealing at 53°C for 15 sec, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The amplification was performed in a LifePro Thermal Cycler (Hangzhou Bioer Technology Co. Ltd., Hangzhou, China) with the following primers: GSEAR-1 5ʹ-GGTTATCAATGTGCGGGTGG-3ʹ and GSEAR-2 5ʹ-CGGCACTTTTTTCTCTTCGG-3ʹ for sea (102bp); HLA-1 5ʹ-CTGATTACTATCCAAGAAATTCGATTG-3ʹ and HLA-2 5ʹ-CTTTCCAGCCTACTTTTTTATCAGT-3ʹ for hla (209bp); HLD-1 5ʹ-AAGAATTTTTATCTTAATTAAGGAAGGAGTG-3ʹ and 5ʹ-TTAGTGAATTTGTTCACTGTGTCGA-3ʹ for hld (111bp); GTSSTR-1 5ʹ-ACCCCTGTTCCCTTATCATC-3ʹ and GTSSTR-2 5ʹ-TTTTCAGTATTTGTAACGCC-3ʹ for tst (326bp); and PVL-1 5ʹ-ATCATTAGGTAAAATGTCTGGACATGATCCA-3ʹ and PVL-2 5ʹ-GCATCAAGTGTATTGGATAGCAAAAGC-3ʹ for pvl (443bp). S. aureus N315 (hla and hld), S. aureus JCSC 4469 (sea and tst), and S. aureus ATCC 14458 (pvl) were included as control strains.

The overall prevalence rates of the virulence genes among the HA-MRSA isolates were as follows: sea: 4.5%; pvl: 18.6%; tst: 27.7%; hla: 87.6%; and hld: 90.4%. The distribution pattern is presented in Figure 1. The respiratory tract (40.7%) and blood (22.6%) were the major MRSA recovery sites, which were followed by the minor sites.

FIGURE 1:
Heatmap showing the distribution of virulence genes among 177 MRSA clinical isolates, according to infection site. sea: staphylococcal enterotoxin type A; hla; alpha-hemolysin; hld; delta-hemolysin; pvl: Panton-Valentine leucocidin; tst: toxic shock syndrome toxin; MRSA: methicillin-resistant Staphylococcus aureus.

In this study, the coexistence of the most prevalent virulence genes, sea, hla, hld, pvl, and tst, was investigated in several MRSA clinical isolates. The most common coexistence was hla + hld (40.7%), followed by tst + hla + hld (26.6%) and pvl + hla + hld (13.6%). The least frequent were hla + hld + sea (1.7%), hla + hld + sea + pvl (1.7%), hla + hld + pvl + tst (0.6%), and hla + hld + sea + tst (0.6%). Despite these low prevalence rates, 49 out of 49 tst-positive isolates and 7 out of 8 sea-positive isolates contained at least one other virulence gene.

Some MRSA isolates (45%) recovered from blood cultures contained both hla and hld, and 92.5% that contained both genes, harbored another gene. In 10 isolates, no virulence gene was found, and no strain contained all genes.

Staphylococcus aureus is a dangerous and versatile pathogen that can cause a multitude of different diseases. Most frequently, it causes infections of the skin and respiratory tract44. Otto M. Staphylococcus aureus toxins. Curr Opin Microbiol. 2014;17:32-7.. S. aureus secretes a group of peptides that can damage the host cell plasma membrane, such as pore-forming toxins like hemolysins99. Vandenesch F, Lina G, Henry T. Staphylococcus aureus hemolysins, bi-component leukocidins, and cytolytic peptides: a redundant arsenal of membrane-damaging virulence factors? Front Cell Infect Microbiol. 2012;16(2):12.. The vast majority of S. aureus strains harbors all four hemolysin genes, hla, hlb, hld, and hlg. It is notable that the two hemolysin genes we tested in this study showed the highest coexistence, highlighting their importance to virulence.

Toxic shock syndrome toxin-1(TSST-1) is a potent superantigen, and it is the most common cause of toxic shock syndrome. It is produced exclusively by S. aureus, and approximately 20% of natural isolates are TSST-1 producers1010. Lindsay JA, Ruzin A, Ross HF, Kurepina N, Novick RP. The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Mol Microbiol. 1998;29(2):527-43.. In this study, 27.7% of the MRSA isolates harbored the tst gene, which is inserted into a pathogenic island1111. Hacker J, Blum-Oehler G, Mühldorfer I, Tschäpe H. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol . 1997;23(6):1089-97., which is widely assumed to be a mobile element. Therefore, TSST-1 can be transmitted through horizontal transfer, which is responsible for its spread among S. aureus strains.

The Panton-Valentine leucocidin (PVL) toxin in S. aureus is responsible for the destruction of polymorphonuclear and mononuclear cells, through necrosis and apoptosis, which usually causes skin or soft tissue infections and necrotizing pneumonia1212. Shallcross LJ, Fragaszy E, Johnson AM, Hayward AC. The role of the Panton-Valentine leucocidin toxin in staphylococcal disease: a systematic review and meta-analysis. Lancet Infect Dis. 2013;13(1):43-54.. In our study, pvl was detected in 18.6% of the HA-MRSA strains, as was observed in previous reports1313. Hu Q, Cheng H, Yuan W, Zeng F, Shang W, Tang D, et al. Panton-Valentine Leukocidin (PVL)-positive health care-associated methicillin-resistant Staphylococcus aureus isolates are associated with skin and soft tissue infections and colonized mainly by infective PVL-encoding bacteriophages. J Clin Microbiol. 2015;53(1):67-72.,1414. Özekinci T, Dal T, Yanık K, Özcan N, Can Ş, Tekin A, et al Panton-Valentine leukocidin in community and hospital-acquired Staphylococcus aureus strains. Biotechnol Biotechnol Equip. 2014;28(6):1089-94.. The increasing prevalence of pvl in HA-MRSA is likely because of the presence of infective PVL phages1313. Hu Q, Cheng H, Yuan W, Zeng F, Shang W, Tang D, et al. Panton-Valentine Leukocidin (PVL)-positive health care-associated methicillin-resistant Staphylococcus aureus isolates are associated with skin and soft tissue infections and colonized mainly by infective PVL-encoding bacteriophages. J Clin Microbiol. 2015;53(1):67-72..

In summary, horizontal transfer of virulence genes is very common in S. aureus, which is suggested by the considerable coexistence of several virulence genes in this study.

Acknowledgments

Adriana M. Rossato was supported by a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We also would like to thank Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) for technical support in the development of this study.

REFERENCES

  • 1
    Händel N, Schuurmans JM, Brul S, ter Kuile BH. Compensation of the metabolic costs of antibiotic resistance by physiological adaptation in Escherichia coli Antimicrob Agents Chemother. 2013;57(8):3752-62.
  • 2
    Tong SYC, Chen LF, Fowler VG. Colonization, pathogenicity, host susceptibility, and therapeutics for Staphylococcus aureus: what is the clinical relevance? Seminars in Immunopathology. 2012;34(2):185-200.
  • 3
    Sollid JU, Furberg AS, Hanssen AM, Johannessen M. Staphylococcus aureus: determinants of human carriage. Infect Genet Evol. 2014;21:531-41.
  • 4
    Otto M. Staphylococcus aureus toxins. Curr Opin Microbiol. 2014;17:32-7.
  • 5
    Yu F, Liu Y, Lv J, Qi X, Lu C, Ding Y. Antimicrobial susceptibility, virulence determinant carriage and molecular characteristics of Staphylococcus aureus isolates associated with skin and soft tissue infections. Braz J Infect Dis. 2015;19(6):614-22.
  • 6
    Kong C, Neoh HM, Nathan S. Targeting Staphylococcus aureus toxins: a potential form of anti-virulence therapy. Toxins (Basel). 2016;8(3):72.
  • 7
    Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: twenty-fourth informational supplement. Document M100-S24. Wayne, PA: CLSI; 2015. p.124-39.
  • 8
    Hoseini Alfatemi SM, Motamedifar M, Hadi N, Sedigh Ebrahim Saraie H. Analysis of virulence genes among methicillin-resistant Staphylococcus aureus (MRSA) Strains. Jundishapur J Microbiol. 2014;7(6):e10741.
  • 9
    Vandenesch F, Lina G, Henry T. Staphylococcus aureus hemolysins, bi-component leukocidins, and cytolytic peptides: a redundant arsenal of membrane-damaging virulence factors? Front Cell Infect Microbiol. 2012;16(2):12.
  • 10
    Lindsay JA, Ruzin A, Ross HF, Kurepina N, Novick RP. The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus Mol Microbiol. 1998;29(2):527-43.
  • 11
    Hacker J, Blum-Oehler G, Mühldorfer I, Tschäpe H. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol . 1997;23(6):1089-97.
  • 12
    Shallcross LJ, Fragaszy E, Johnson AM, Hayward AC. The role of the Panton-Valentine leucocidin toxin in staphylococcal disease: a systematic review and meta-analysis. Lancet Infect Dis. 2013;13(1):43-54.
  • 13
    Hu Q, Cheng H, Yuan W, Zeng F, Shang W, Tang D, et al. Panton-Valentine Leukocidin (PVL)-positive health care-associated methicillin-resistant Staphylococcus aureus isolates are associated with skin and soft tissue infections and colonized mainly by infective PVL-encoding bacteriophages. J Clin Microbiol. 2015;53(1):67-72.
  • 14
    Özekinci T, Dal T, Yanık K, Özcan N, Can Ş, Tekin A, et al Panton-Valentine leukocidin in community and hospital-acquired Staphylococcus aureus strains. Biotechnol Biotechnol Equip. 2014;28(6):1089-94.

  • Financial support: This study was supported by Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS).

Publication Dates

  • Publication in this collection
    May-Jun 2018

History

  • Received
    24 Aug 2017
  • Accepted
    07 Dec 2017
Sociedade Brasileira de Medicina Tropical - SBMT Caixa Postal 118, 38001-970 Uberaba MG Brazil, Tel.: +55 34 3318-5255 / +55 34 3318-5636/ +55 34 3318-5287, http://rsbmt.org.br/ - Uberaba - MG - Brazil
E-mail: rsbmt@uftm.edu.br