Sir,
Staphylococcus aureus produces many virulence factor toxins and exoenzymes, including exfoliative toxin, enterotoxin, toxic shock syndrome toxin (TSST), haemolysins, proteases, lipases and nucleases. Current advances in the study of S. aureus have shown that many regulatory elements for bacterial signal transduction and gene expression, as represented by two-component systems (TCSs) and the Sar family, respectively, control the expression of virulence factors.1 Genome-wide analysis of several S. aureus strains revealed that 16 types of TCSs are encoded in chromosomal DNA. Besides the influence of TCSs on the expression of virulence factors, some TCSs, including vra and aps/gra, have recently been demonstrated to be associated with susceptibility to antibacterial agents.2,3 However, the effects of these TCSs on susceptibility to antibacterial agents has never been compared in order to establish which TCSs play a role in susceptibility to antibacterial agents. Therefore, in this study, a battery of TCS-inactivated mutant strains of S. aureus MW2 were analysed to evaluate their possible involvement in the regulation of susceptibility to several antibacterial agents.
We attempted to construct insertionally inactivated mutants for 16 different TCSs in the community-acquired methicillin-resistant S. aureus MW2 strain by pCL52.1 (a thermosensitive Escherichia coli–S. aureus shuttle vector) integration as described previously.4 The target gene was selected as a forward open reading frame (ORF) in an operon that includes two ORFs encoding sensor and regulatory elements. S. aureus was grown in trypticase soy broth (TSB) (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). Tetracycline (10 mg/L) was added when necessary.
The MICs of various antibacterial agents were determined using the microdilution method described previously.5 Oxacillin, bacitracin and vancomycin (Sigma Chemical Co., Ltd, St Louis, MO, USA) and teicoplanin (Sigma–Aldrich, Tokyo, Japan), as well as gentamicin, erythromycin, ofloxacin, benzalkonium chloride and chlorhexidine gluconate (Wako Pure Chemical Industries, Ltd, Osaka, Japan), were used. The MBC, which gives a killing rate >99.9%, was determined to evaluate the antibacterial activity of the antimicrobial peptides human β-defensin-3 (hBD3) and LL-37, following the protocols described previously.5
We were able to construct all but one TCS mutant, termed VicRK (Table 1), because vicRK was reported as an essential gene in cell wall metabolism in S. aureus. Table 1 shows the MICs of five different classes of antibacterial agents, including: (i) cell wall inhibitors (β-lactams; oxacillin, vancomycin, teicoplanin and bacitracin); (ii) protein synthesis inhibitors (erythromycin and gentamicin); (iii) a DNA synthesis inhibitor (ofloxacin); (iv) disinfectants (chlorhexidine gluconate and benzalkonium chloride) (data not shown); and (v) antimicrobial peptides (hBD3 and LL-37) against 15 TCS mutants of the S. aureus MW2 strain.
Table 1.
Susceptibility of S. aureus TCS mutants to various antibacterial agents
| Inactivated gene |
MIC (mg/L) |
MBC (mg/L) |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Strain | ORF no.a | Gene nameb | OXA | VAN | TEC | BAC | GEN | ERY | OFX | hBD3 | LL37 |
| MW2 | wild-type | — | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK61 | MW0199 | unassigned (HK) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK62 | MW0236 | lytS (HK) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK64 | MW0621 | apsR (RR) | 4 | 2 | 0.5 | 64 | 1 | 0.25 | 1 | 4 | 2 |
| FK65 | MW0668 | saeR (RR) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK66 | MW1208 | unassigned (HK) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK67 | MW1305 | arlR (RR) | 2 | 2 | 0.5 | 64 | 2 | 0.25 | 1 | 2 | 1 |
| FK68 | MW1446 | srrA (RR) | 16 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK69 | MW1637 | phoP (RR) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK71 | MW1790 | unassigned (HK) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK72 | MW1825 | vraS (HK) | 2 | 1 | 0.25 | 64 | 2 | 0.25 | 1 | 8 | 4 |
| FK73 | MW1962 | agrC (HK) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK74 | MW2002 | kdpD (HK) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK75 | MW2282 | hssR (RR) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK76 | MW2314 | nreC (HK) | 8 | 2 | 0.5 | 64 | 4 | 0.25 | 1 | 8 | 4 |
| FK77 | MW2545 | unassigned (HK) | 8 | 2 | 0.5 | 16 | 4 | 0.25 | 1 | 8 | 4 |
OXA, oxacillin; VAN, vancomycin; TEC, teicoplanin; BAC, bacitracin; GEN, gentamicin; ERY, erythromycin; OFX, ofloxacin.
aGene ID in S. aureus MW2.
bHK, histidine kinase; RR, response regulator.
The MICs of oxacillin and gentamicin and the MBCs of hBD3 and LL-37 against aps/gra (FK64) and arl (FK67) mutants were decreased, although the MIC and MBC values were different between the two strains. The MICs of oxacillin, vancomycin, teicoplanin and gentamicin for the vra mutant (FK72) were decreased. The mutation of one uncharacterized TCS (FK77) decreased the MIC of bacitracin, while the MICs of other antibiotic agents tested were not altered. The MIC of oxacillin for the srr mutant (FK68) was increased by 2-fold, suggesting that srr may play a down-regulatory role in bacterial resistance to oxacillin. No mutations altered the MICs of the two disinfectants.
Among the TCSs tested in this study, vra and aps/gra have already been reported to affect the susceptibilities to some antibacterial agents, while arl, srr and one uncharacterized TCS were not reported previously. Interestingly, several TCSs affected susceptibility to oxacillin. It is well known that clinically isolated methicillin-resistant S. aureus (MRSA) strains show a variety of β-lactam resistance levels, from low to high. Many factors are known to affect the β-lactam resistance level in MRSA strains, but the precise underlying mechanisms are still not clearly elucidated.6 Our study shows that multiple TCSs might be associated with the variety of β-lactam resistance levels observed in clinical strains of MRSA.
Funding
This work was supported by a grant-in-aid for scientific research on Health and Labor Sciences Research Grants from the Ministry of Health and Welfare of Japan and an NIDCR grant DE-18310.
Transparency declarations
None to declare.
References
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