Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-013-1987-5
ARTICLE
Association between susceptibility to photodynamic oxidation and the genetic background of Staphylococcus aureus A. Rapacka-Zdonczyk & A. Rhod Larsen & J. Empel & A. Patel & M. Grinholc
Received: 30 July 2013 / Accepted: 18 September 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Multidrug resistant strains of Staphylococcus aureus are a major cause of skin and soft tissue infections requiring the development of novel and alternative therapeutic options. Photodynamic oxidation is the cornerstone of antimicrobial photodynamic therapy (aPDT) involving the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of oxidizing biological molecules and leads to inactivation of target cells. We have previously shown that susceptibility to aPDT differs significantly across S. aureus isolates and could be associated with several genetic elements. However, the effect of the photodynamic process regarding the S. aureus genetic background has never been reported. We have compared the genetic backgrounds of the strains (SCCmec types, spa types and main clonal complexes) with respect to their susceptibility to protoporphyrin IX-mediated photodynamic inactivation. SCCmec typing revealed no differences in response to photoinactivation. However, detection of spa types and clonal complexes clustered the studied population of MRSA strains Electronic supplementary material The online version of this article (doi:10.1007/s10096-013-1987-5) contains supplementary material, which is available to authorized users A. Rapacka-Zdonczyk : M. Grinholc (*) Laboratory of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland e-mail: [email protected] A. R. Larsen Staphylococcus Laboratory, Statens Serum Institute, 2300 Copenhagen, Denmark J. Empel Department of Molecular Microbiology, National Medicines Institute, Warsaw, Poland A. Patel Centre for Clinical Infection and Diagnostics Research, Department of Infectious Diseases, St Thomas’ Hospital, London, UK
according to their response to photodynamic oxidation. Clonal complex 1 (CC1) accounted for elevated resistance and CC30 (ST36) for susceptibility to photoinactivation. Moreover, spa typing identified isolates resistant (t032) and susceptible to photodynamic oxidation (t051, t015). The very tight association between clonal lineages and response to photodynamic inactivation indicates the important role of genetic background for aPDT efficacy. These results make a case for the development of a diagnostic tool with the predictive value of aPDT efficacy according to an identified genetic background of S. aureus isolates.
Introduction Healthcare-associated infections, especially those caused by methicillin-resistant Staphylococcus aureus strains (HAMRSA), are a great danger to both hospitalized and immunocompromised patients, in whom the organism causes high morbidity and mortality [1]. S. aureus displays extensive genetic variability. Genotyping analyses, such as multilocus sequence typing (MLST) and protein A (spa) sequence typing, demonstrated that the S. aureus population structure is highly clonal with six major clonal complexes (CCs) (CC1, CC5, CC8, CC22, CC30, and CC45) [2–4]. Most mobile genetic elements can readily spread horizontally among S. aureus strains of the same clonal cluster, while transfer between clusters is limited. S. aureus strains are generally classified into two classes according to their resistance to β-lactam antibiotics: methicillin-sensitive S. aureus (MSSA) and MRSA strains. One of the resistance mechanisms to β-lactams used by MRSA is based on the production of an alternative penicillin-binding protein (PBP) encoded by the mecA gene; this protein is termed PBP2a. In MRSA, exposure to β-lactams inactivates the four high-binding-affinity PBPs normally present. PBP2a, which displays a low affinity for βlactams, takes over the functions of these PBPs, permitting the cell to grow. The mecA gene is located on a mobile genetic
Eur J Clin Microbiol Infect Dis
element integrated into the bacterial chromosome and called the staphylococcal cassette chromosome mec (SCCmec) [5]. Eleven different types of SCCmec, assigned SCCmec I to SCCmec XI, have been defined so far. Most epidemiological investigations focus on types I to VI, and studies on SCCmec types VII to XI are rarely reported [6–9]. This genetic element is widely used for the determination of MRSA clones. Identification of SCCmec type can be used to predict strain origin, as the majority of nosocomial isolates harbor SCCmec I, II, or III, whereas community-associated MRSA strains (CA-MRSA) are characterized by SCCmec IV and V [8–10]. Furthermore, it seems that SCCmec element is involved in staphylococcal virulence. It has been shown that SCCmec type-associated virulence factors substantially influence the progression of staphylococcal bacteremia, and SCCmec II S. aureus strains cause the highest morbidity among hospitalized patients [11]. Thus, the determination of SCCmec types, clonal complexes, or spa types may have an important diagnostic value. Whilst no single typing method meets all the requirements for typing techniques, there have been recent trends toward the use of spa, MLST, and SCCmec methods [12]. Large typing databases and networks have been developed to handle the data. The spa typing method is based on sequencing of the polymorphic X region of the protein A gene (spa), present in all strains of S. aureus. The X region consists of a variable number of 24-bp repeats flanked by well-conserved regions. This single-locus sequence-based typing method combines a number of technical advantages, such as rapidity, reproducibility, and portability. Moreover, because of its repeat structure, the spa locus simultaneously indexes micro- and macrovariations, enabling the use of spa typing in both local and global epidemiological studies [13]. The current terminology to describe a lineage is based on multilocus sequence typing (MLST) clonal complexes (CCs). MLST involves sequencing seven housekeeping genes and assigning a sequence type (ST) number to isolates that are identical [12]. MLST is recommended for use in regional laboratories and is a useful tool for defining lineages and scientific analysis. These methods, plus SCCmec, provide the minimum requirements for appropriate molecular epidemiology [12]. The antimicrobial photodynamic therapy (aPDT) has gained increasing importance because of its ability to eradicate a broad spectrum of pathogenic microorganisms [14, 15]. In the clinical area it could be a potential alternative to the antibiotics used to treat staphylococcal infections. The photodynamic process involves the combined use of light, molecular oxygen, and a photosensitive compound (photosensitizer) [16]. This interaction leads to production of reactive oxygen species and free radicals responsible for oxidation of many biological molecules (enzymes, proteins, lipids, and nucleic acids), leading to cell death [17]. Additionally, the resistance mechanisms towards photodynamic inactivation have not yet been observed [18, 19]. However, our studies of the photoinactivation (PDI) of multiresistant pathogenic bacteria
have shown that the effectiveness of PDI is strain-dependent and that one should search for possible strategies to overcome resistant phenotype, but, on other hand, to develop the diagnostic tool clustering strains according to their susceptibility to PDI [20–24]. The mechanism underlying this phenomenon is still poorly understood. Thus, by applying SCCmec typing and collecting the isolates representing different spa genotypes and clonal clusters, we aimed, in particular, to assess the contribution of the bacterial genetic background in S. aureus response to photodynamic oxidation.
Materials and methods Bacterial isolates In total, 438S. aureus strains (418 MRSA and 20 MSSA) were used. Of them, 321 clinical MRSA strains were isolated between 2002 and 2012 at the Provincial Hospital in Gdansk and Hospital in Koszalin (Poland) and were previously characterized. The isolates were characterized by Gram staining and the ability to produce coagulase and the clumping factor using Slidex Staph Plus (bioMèrieux, France) and were reidentified to the species level using the MALDI-TOF MS (Bruker Daltonics, Germany). Methicillin resistance was determined using a disc-diffusion method as well as a latex test detecting PBP2a protein (Staphytect Plus; Oxoid, Lenexa,KS, USA) [25]. Of the 321 strains collected, 40.5 % (130 isolates) were isolated from surveillance cultures, 46.4 % (149 isolates) from patients with local infections, 11.8 % (38 isolates) from patients with bacteremia and generalized infections and 1.3 % (4 isolates) from infections connected with endoprostheses. An additional 97 MRSA and 20 MSSA isolates, with identified CCs and spa type were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA), Staphylococcus Laboratory, Statens Serum Institute in Denmark, Centre for Clinical Infection and Diagnostics Research in the UK, University of Oviedo in Spain, and National Medicines Institute in Poland. The strains are listed in Online Resource 1. Clonal complexes (CC) were determined with the use of MLST. All clonal complexes, excluding CC8, were represented by single ST. CC8 included strains with ST8 and ST247. Additionally, the reference strains of S. aureus listed in Table 1 were used to optimize SCCmec type determination. The abundance of S. aureus isolates in each group is provided in Table 2. The molecular characteristics of isolates are provided in Table 3. MALDI-TOF MS Three hundred and twenty-one S . aureus clinical strains isolated from hospitals in Poland were examined by MALDITOF MS using a Microflex LT instrument (Bruker Daltonics,
Eur J Clin Microbiol Infect Dis Table 1 Primers and reference strains used in the study Strain used as standard
Primer
Oligonucleotide sequence (5′–3′)
Size (bp)
Reference
NCTC 10442
SCC I
613
[27]
N315
SCC II
287
[27]
85/2082
SCC III
243
[27]
8/6-3P
SCC IVb
1,000
[27]
81-108
SCC IVc
677
[27]
JSCS 4469
SCC IVd
1,242
[27]
WIS
SCC V
325
[27]
NCTC 10442
Sa442
108
[27]
NCTC 10442
mecA
Forward: GCT TTA AAG AGT GTC GTT ACA GG Reverse: GTT CTC TCA TAG TAT GAC GTC C Forward: GAT TAC TTC AGA ACC AGG TCA T Reverse: TAA ACT GTG TCA CAC GAT CCA T Forward: CAT TTG TGA AAC ACA GTA CG Reverse: GTT ATT GAG ACT CCT AAA GC Forward: AGT ACA TTT TAT CTT TGC GTA Reverse: AGT CAT CTT CAA TAT GGA GAA AGT A Forward: TCT ATT CAA TCG TTC TCG TAT T Reverse: TCG TTG TCA TTT AAT TCT GAA CT Forward: AAT TCA CCC GTA CCT GAG AA Reverse: AGA ATG TGG TTA TAA GAT AGC TA Forward: GAA CAT TGT TAC TTA AAT GAG CG Reverse: TGA AAG TTG TAC CCT TGA CAC C Forward: AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG Reverse: CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA Forward: TCC AGA TTA CAA CTT CAC CAG G Reverse: CCA CTT CAT ATC TTG TAA CG
162
[27]
Leipzig, Germany), Flexcontrol 3.0 software and the Biotyper 2.0 database (Bruker Daltonics, Germany). The data analysis was performed according to the manufacturer’s instructions. The samples were covered with 1 ml matrix solution (a Table 2 Distribution of methicillin-resistant Staphylococcus aureus (MRSA) isolates by typing procedures
Typing procedures Clonal complex CC1 CC5 CC8 CC22 CC30 CC45 SCCmec I II III IV V spa type t002 t008 t015 t026 t032
a
Only types represented by more than three isolates were included (all types are listed in Online Resource 1)
t051 t064 t230 Othera
Number (%) of isolates
5 (5.15) 22 (22.65) 25 (25.80) 8 (8.25) 3 (3.10) 34 (35.05) 31 (7.40) 222 (53.10) 22 (5.25) 124 (29.70) 19 (4.55) 15 (15.50) 7 (7.20) 12 (12.40) 4 (4.10) 6 (6.20)
saturated solution of a-cyano-4-hydroxycinnamic acid in 50 % acetonitrile, 2.5 % trifluoroacetic acid). The analyzed mass range of spectra was 2,000–20,000 m/z. Each spectrum was obtained after 240 shots in an automatic acquisition mode. For the identification approach, a mass-to-charge range of 3,000–15,000 Da was used. Identification was performed in duplicate and the higher score was retained. The identification score cut-off values were applied on each measurement according to the manufacturer’s instructions. According to this score system, a score of
ARTICLE
Association between susceptibility to photodynamic oxidation and the genetic background of Staphylococcus aureus A. Rapacka-Zdonczyk & A. Rhod Larsen & J. Empel & A. Patel & M. Grinholc
Received: 30 July 2013 / Accepted: 18 September 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Multidrug resistant strains of Staphylococcus aureus are a major cause of skin and soft tissue infections requiring the development of novel and alternative therapeutic options. Photodynamic oxidation is the cornerstone of antimicrobial photodynamic therapy (aPDT) involving the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of oxidizing biological molecules and leads to inactivation of target cells. We have previously shown that susceptibility to aPDT differs significantly across S. aureus isolates and could be associated with several genetic elements. However, the effect of the photodynamic process regarding the S. aureus genetic background has never been reported. We have compared the genetic backgrounds of the strains (SCCmec types, spa types and main clonal complexes) with respect to their susceptibility to protoporphyrin IX-mediated photodynamic inactivation. SCCmec typing revealed no differences in response to photoinactivation. However, detection of spa types and clonal complexes clustered the studied population of MRSA strains Electronic supplementary material The online version of this article (doi:10.1007/s10096-013-1987-5) contains supplementary material, which is available to authorized users A. Rapacka-Zdonczyk : M. Grinholc (*) Laboratory of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland e-mail: [email protected] A. R. Larsen Staphylococcus Laboratory, Statens Serum Institute, 2300 Copenhagen, Denmark J. Empel Department of Molecular Microbiology, National Medicines Institute, Warsaw, Poland A. Patel Centre for Clinical Infection and Diagnostics Research, Department of Infectious Diseases, St Thomas’ Hospital, London, UK
according to their response to photodynamic oxidation. Clonal complex 1 (CC1) accounted for elevated resistance and CC30 (ST36) for susceptibility to photoinactivation. Moreover, spa typing identified isolates resistant (t032) and susceptible to photodynamic oxidation (t051, t015). The very tight association between clonal lineages and response to photodynamic inactivation indicates the important role of genetic background for aPDT efficacy. These results make a case for the development of a diagnostic tool with the predictive value of aPDT efficacy according to an identified genetic background of S. aureus isolates.
Introduction Healthcare-associated infections, especially those caused by methicillin-resistant Staphylococcus aureus strains (HAMRSA), are a great danger to both hospitalized and immunocompromised patients, in whom the organism causes high morbidity and mortality [1]. S. aureus displays extensive genetic variability. Genotyping analyses, such as multilocus sequence typing (MLST) and protein A (spa) sequence typing, demonstrated that the S. aureus population structure is highly clonal with six major clonal complexes (CCs) (CC1, CC5, CC8, CC22, CC30, and CC45) [2–4]. Most mobile genetic elements can readily spread horizontally among S. aureus strains of the same clonal cluster, while transfer between clusters is limited. S. aureus strains are generally classified into two classes according to their resistance to β-lactam antibiotics: methicillin-sensitive S. aureus (MSSA) and MRSA strains. One of the resistance mechanisms to β-lactams used by MRSA is based on the production of an alternative penicillin-binding protein (PBP) encoded by the mecA gene; this protein is termed PBP2a. In MRSA, exposure to β-lactams inactivates the four high-binding-affinity PBPs normally present. PBP2a, which displays a low affinity for βlactams, takes over the functions of these PBPs, permitting the cell to grow. The mecA gene is located on a mobile genetic
Eur J Clin Microbiol Infect Dis
element integrated into the bacterial chromosome and called the staphylococcal cassette chromosome mec (SCCmec) [5]. Eleven different types of SCCmec, assigned SCCmec I to SCCmec XI, have been defined so far. Most epidemiological investigations focus on types I to VI, and studies on SCCmec types VII to XI are rarely reported [6–9]. This genetic element is widely used for the determination of MRSA clones. Identification of SCCmec type can be used to predict strain origin, as the majority of nosocomial isolates harbor SCCmec I, II, or III, whereas community-associated MRSA strains (CA-MRSA) are characterized by SCCmec IV and V [8–10]. Furthermore, it seems that SCCmec element is involved in staphylococcal virulence. It has been shown that SCCmec type-associated virulence factors substantially influence the progression of staphylococcal bacteremia, and SCCmec II S. aureus strains cause the highest morbidity among hospitalized patients [11]. Thus, the determination of SCCmec types, clonal complexes, or spa types may have an important diagnostic value. Whilst no single typing method meets all the requirements for typing techniques, there have been recent trends toward the use of spa, MLST, and SCCmec methods [12]. Large typing databases and networks have been developed to handle the data. The spa typing method is based on sequencing of the polymorphic X region of the protein A gene (spa), present in all strains of S. aureus. The X region consists of a variable number of 24-bp repeats flanked by well-conserved regions. This single-locus sequence-based typing method combines a number of technical advantages, such as rapidity, reproducibility, and portability. Moreover, because of its repeat structure, the spa locus simultaneously indexes micro- and macrovariations, enabling the use of spa typing in both local and global epidemiological studies [13]. The current terminology to describe a lineage is based on multilocus sequence typing (MLST) clonal complexes (CCs). MLST involves sequencing seven housekeeping genes and assigning a sequence type (ST) number to isolates that are identical [12]. MLST is recommended for use in regional laboratories and is a useful tool for defining lineages and scientific analysis. These methods, plus SCCmec, provide the minimum requirements for appropriate molecular epidemiology [12]. The antimicrobial photodynamic therapy (aPDT) has gained increasing importance because of its ability to eradicate a broad spectrum of pathogenic microorganisms [14, 15]. In the clinical area it could be a potential alternative to the antibiotics used to treat staphylococcal infections. The photodynamic process involves the combined use of light, molecular oxygen, and a photosensitive compound (photosensitizer) [16]. This interaction leads to production of reactive oxygen species and free radicals responsible for oxidation of many biological molecules (enzymes, proteins, lipids, and nucleic acids), leading to cell death [17]. Additionally, the resistance mechanisms towards photodynamic inactivation have not yet been observed [18, 19]. However, our studies of the photoinactivation (PDI) of multiresistant pathogenic bacteria
have shown that the effectiveness of PDI is strain-dependent and that one should search for possible strategies to overcome resistant phenotype, but, on other hand, to develop the diagnostic tool clustering strains according to their susceptibility to PDI [20–24]. The mechanism underlying this phenomenon is still poorly understood. Thus, by applying SCCmec typing and collecting the isolates representing different spa genotypes and clonal clusters, we aimed, in particular, to assess the contribution of the bacterial genetic background in S. aureus response to photodynamic oxidation.
Materials and methods Bacterial isolates In total, 438S. aureus strains (418 MRSA and 20 MSSA) were used. Of them, 321 clinical MRSA strains were isolated between 2002 and 2012 at the Provincial Hospital in Gdansk and Hospital in Koszalin (Poland) and were previously characterized. The isolates were characterized by Gram staining and the ability to produce coagulase and the clumping factor using Slidex Staph Plus (bioMèrieux, France) and were reidentified to the species level using the MALDI-TOF MS (Bruker Daltonics, Germany). Methicillin resistance was determined using a disc-diffusion method as well as a latex test detecting PBP2a protein (Staphytect Plus; Oxoid, Lenexa,KS, USA) [25]. Of the 321 strains collected, 40.5 % (130 isolates) were isolated from surveillance cultures, 46.4 % (149 isolates) from patients with local infections, 11.8 % (38 isolates) from patients with bacteremia and generalized infections and 1.3 % (4 isolates) from infections connected with endoprostheses. An additional 97 MRSA and 20 MSSA isolates, with identified CCs and spa type were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA), Staphylococcus Laboratory, Statens Serum Institute in Denmark, Centre for Clinical Infection and Diagnostics Research in the UK, University of Oviedo in Spain, and National Medicines Institute in Poland. The strains are listed in Online Resource 1. Clonal complexes (CC) were determined with the use of MLST. All clonal complexes, excluding CC8, were represented by single ST. CC8 included strains with ST8 and ST247. Additionally, the reference strains of S. aureus listed in Table 1 were used to optimize SCCmec type determination. The abundance of S. aureus isolates in each group is provided in Table 2. The molecular characteristics of isolates are provided in Table 3. MALDI-TOF MS Three hundred and twenty-one S . aureus clinical strains isolated from hospitals in Poland were examined by MALDITOF MS using a Microflex LT instrument (Bruker Daltonics,
Eur J Clin Microbiol Infect Dis Table 1 Primers and reference strains used in the study Strain used as standard
Primer
Oligonucleotide sequence (5′–3′)
Size (bp)
Reference
NCTC 10442
SCC I
613
[27]
N315
SCC II
287
[27]
85/2082
SCC III
243
[27]
8/6-3P
SCC IVb
1,000
[27]
81-108
SCC IVc
677
[27]
JSCS 4469
SCC IVd
1,242
[27]
WIS
SCC V
325
[27]
NCTC 10442
Sa442
108
[27]
NCTC 10442
mecA
Forward: GCT TTA AAG AGT GTC GTT ACA GG Reverse: GTT CTC TCA TAG TAT GAC GTC C Forward: GAT TAC TTC AGA ACC AGG TCA T Reverse: TAA ACT GTG TCA CAC GAT CCA T Forward: CAT TTG TGA AAC ACA GTA CG Reverse: GTT ATT GAG ACT CCT AAA GC Forward: AGT ACA TTT TAT CTT TGC GTA Reverse: AGT CAT CTT CAA TAT GGA GAA AGT A Forward: TCT ATT CAA TCG TTC TCG TAT T Reverse: TCG TTG TCA TTT AAT TCT GAA CT Forward: AAT TCA CCC GTA CCT GAG AA Reverse: AGA ATG TGG TTA TAA GAT AGC TA Forward: GAA CAT TGT TAC TTA AAT GAG CG Reverse: TGA AAG TTG TAC CCT TGA CAC C Forward: AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG Reverse: CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA Forward: TCC AGA TTA CAA CTT CAC CAG G Reverse: CCA CTT CAT ATC TTG TAA CG
162
[27]
Leipzig, Germany), Flexcontrol 3.0 software and the Biotyper 2.0 database (Bruker Daltonics, Germany). The data analysis was performed according to the manufacturer’s instructions. The samples were covered with 1 ml matrix solution (a Table 2 Distribution of methicillin-resistant Staphylococcus aureus (MRSA) isolates by typing procedures
Typing procedures Clonal complex CC1 CC5 CC8 CC22 CC30 CC45 SCCmec I II III IV V spa type t002 t008 t015 t026 t032
a
Only types represented by more than three isolates were included (all types are listed in Online Resource 1)
t051 t064 t230 Othera
Number (%) of isolates
5 (5.15) 22 (22.65) 25 (25.80) 8 (8.25) 3 (3.10) 34 (35.05) 31 (7.40) 222 (53.10) 22 (5.25) 124 (29.70) 19 (4.55) 15 (15.50) 7 (7.20) 12 (12.40) 4 (4.10) 6 (6.20)
saturated solution of a-cyano-4-hydroxycinnamic acid in 50 % acetonitrile, 2.5 % trifluoroacetic acid). The analyzed mass range of spectra was 2,000–20,000 m/z. Each spectrum was obtained after 240 shots in an automatic acquisition mode. For the identification approach, a mass-to-charge range of 3,000–15,000 Da was used. Identification was performed in duplicate and the higher score was retained. The identification score cut-off values were applied on each measurement according to the manufacturer’s instructions. According to this score system, a score of
