Reduced expression of the atl autolysin gene and susceptibility to autolysis in clinical heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) and GISA strains

Mandy Wootton1,2,*, Peter M. Bennett1, Alasdair P. MacGowan2 and Timothy R. Walsh1

Bristol Centre for Antimicrobial Research and Evaluation, 1 Department of Cellular and Molecular Medicine, Medical Sciences, University of Bristol, Bristol BS1 8TD, UK; and 2 North Bristol Healthcare Trust, Southmead Hospital, Bristol BS10 5NB, UK


* Corresponding author. Tel: +44-117-928-7526; Fax: +44-117-928-7896; E-mail: mandy.wootton{at}bristol.ac.uk

Received 24 May 2005; returned 3 June 2005; revised 25 July 2005; accepted 26 July 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To assess a link between resistance to Triton X-100 induced autolysis (TIA) and lowered atl expression in a collection of clinical glycopeptide-intermediate Staphylococcus aureus (GISA) and heterogeneous GISA (hGISA).

Methods: Nine clinical GISA, 11 hGISA and 11 glycopeptide-susceptible S. aureus (GSSA), including three pairs of related isolates, were analysed using TIA assays. Lysostaphin MICs were determined by a broth microdilution technique and reverse transcriptase PCR was used to compare atl expression levels in all isolates.

Results: Eight of nine clinical GISA and six of 11 hGISA exhibited lower susceptibility to TIA and higher MICs of lysostaphin than GSSA. Eight of nine GISA and all hGISA strains had lowered atl expression levels compared with GSSA.

Conclusions: The majority of GISA and hGISA isolates exhibited lowered susceptibility to TIA and lysostaphin and reduced atl expression when compared with GSSA isolates. These factors could contribute to, or predispose to the development of, a thickened cell wall and glycopeptide-intermediate resistance.

Keywords: antibiotic resistance , cell wall , mechanism of resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Glycopeptide-intermediate-resistant Staphylococcus aureus (GISA) and heterogeneous GISA (hGISA) strains share the common characteristic of a thickened cell wall. The role of the thickened cell wall in the mechanism of glycopeptide resistance is uncertain, as is the precise mechanism of cell wall thickening. However, it is possible that reduced expression of murein hydrolases, including Atl, may play a role. In two recent studies, laboratory-derived GISA and four clinical GISA strains exhibited lowered susceptibility to Triton X-100 induced autolysis (TIA).1,2 The laboratory-generated GISA also showed reduced hydrolase activity, but no such change was seen in clinical GISA.1,2 To investigate this further, we examined expression levels of the major autolysin gene, atl, autolytic activity and susceptibility to lysostaphin in a number of clinical GISA, hGISA and glycopeptide-susceptible S. aureus (GSSA) isolates.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

Glycopeptide susceptibility was determined by population analysis profile–area under curve (PAP-AUC) using the criteria: <0.9, GSSA; 0.9–1.29, hGISA; and ≥1.3, GISA.3 Expression of atl and susceptibility to TIA and lysostaphin were studied in nine clinical GISA (isolates MI, NJ, GL3700, GL2759, SW307, SL, PC3, LIM3 and Mu50), 11 clinical hGISA (isolates Fduf, AGN, SH23, NW1018, SW309, SL6096, SMH2, LIM1, PC1, LLE and Mu3) and 11 clinical GSSA (randomly selected on the basis of glycopeptide susceptibility; from the UK, including EMRSA-15 and EMRSA-16 clonal types). This collection included three pairs of related isolates (LLA-GSSA/LLE-hGISA, PC1-hGISA/PC3-GISA and LIM1-hGISA/LIM3-GISA). LLA and LLE were clonal strains isolated from an 82-year-old male with chronic renal failure; LLA prior to vancomycin therapy and LLE after 22 days of vancomycin.

Autolytic assays

Cultures were grown in brain–heart infusion broth (BBL, Cockeysville, MD, USA) and harvested at mid-log phase (OD600 0.7). Cells were pelleted, washed in ice-cold water, then resuspended in 0.05% Triton X-100. Optical density at 600 nm was read at time 0 and at 30 min intervals. Percentage lysis was calculated by dividing OD by initial OD x 100 and an AUC was calculated. A Kruskal–Wallis statistical test was performed on AUC data.

Lysostaphin MIC determination

Exponentially growing cells were transferred to tryptone soya broth (TSB) (Oxoid, Basingstoke, UK) containing serial two-fold dilutions of lysostaphin (range 0.06–64 mg/L; Sigma, Poole, UK) in round-bottomed microtitre wells. Plates were incubated at 37°C for 16 h and the MIC was taken as the lowest concentration to inhibit all cell growth.

atl gene expression: reverse transcriptase (RT)–PCR and atl sequencing

RNA was recovered from exponentially-growing cells in TSB (OD600 0.7) using a dedicated kit (Qiagen Rneasy Mini 74104) and stored at –20°C. DNA was removed from RNA extractions using DNase (according to the manufacturer's instructions) and RNA concentration was quantified by spectrophotometry (Promega, USA). One microgram of RNA was used per RT–PCR (Qiagen One-step RT-PCR Kit, 210210, USA) together with gene-specific primers (atl-F: 5'-CAGTTAGCAAGATTGCTCAAG-3', atl-R: 5'-CCGTTACCTGTTTCTAATAGG-3', atl-promoter F: 5'-GGAAGGCATCGAGCAT-3', atl-promoter R: 5'-GCGTTAATGCAACCAT-3'). Expression levels of atl in GISA were evaluated in triplicate on agarose gels, using expression levels in GSSA as equivalent to normal expression. Control amplifications, using 16S rRNA primers, were performed on every isolate to eliminate artefactual expression differences resulting from different template concentrations. Sequence analysis of the atl promoter region was determined by the Advanced Biotechnology Centre (Imperial College School of Medicine, London, UK) and compared using DNASTAR-SeqMan 5.0 software (DNASTAR Inc., USA).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
All GISA, except Mu50, showed reduced susceptibility to TIA in comparison with hGISA and GSSA isolates. Figure 1(a) shows mean autolysis profile AUCs from GISA, hGISA and GSSA. Susceptibility to TIA decreased with increasing glycopeptide-intermediate resistance, with GISA strains exhibiting the lowest susceptibility, followed by hGISA and GSSA isolates (means were significantly different; P = 0.02). The reduced susceptibility to TIA in clinical GISA and hGISA, but not in Mu50 or Mu3, agrees with data presented in other studies,1,2 where it was suggested that lowered susceptibility enhanced the capability of S. aureus to evade the lysis-inducing effect of vancomycin. In clinically related pairs of isolates, resistance to TIA, indicated by AUC, was also consistently higher in the more glycopeptide intermediately resistant isolates (Figure 1b).



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Figure 1. Autolysis assays showing (a) mean % absorbance in GISA, hGISA and GSSA strains, (b) AUC (calculated using GraphPad Prism®) in related strains sets (PAP-AUCs: LLA-0.65, LLE-1.14, PC1-1.21, PC3-1.43, LIM1-1.14, LIM3-1.4) and (c) frequency of lysostaphin MICs in GISA, hGISA and GSSA. GISA and hGISA AUCs are calculated without Mu50 and Mu3.

 
MICs of lysostaphin revealed a trend towards higher resistance among GISA isolates compared with hGISA and GSSA isolates (Figure 1c). Lysostaphin resistance in S. aureus is associated with the genes epr and femAB, the former responsible for increasing serine content and decreasing glycine content of the peptidoglycan interpeptide bridge and the latter for shortened glycine bridges.4 However, evidence suggests that the peptidoglycan of clinical and laboratory-generated GISA isolates has glycine bridges of normal length and composition.5,6

Eight of nine GISA and all 11 hGISA exhibited lower atl expression, as indicated by less intense bands on agarose gels, when compared with atl expression in all GSSA isolates, which suggests a correlation with glycopeptide resistance. Expression of atl in all isolates of the related pairs (including the GSSA, LLA; Figure 2) appeared lower than in control GSSA isolates. For pair LLA and LLE, atl expression appeared to be greater in the hGISA isolate, the reasons for which are unknown. The lower atl expression exhibited by LLA may indicate that reduction in atl expression takes place prior to development of glycopeptide-intermediate resistance and therefore that it could be a predisposing factor of the GISA/hGISA phenotype. Previous investigations into atl have reported similar sequences in glycopeptide-resistant and susceptible strains, although expression rates could be altered by a mutated promoter region.1 A 250 bp region upstream of atl was sequenced and compared in all isolates and found to be identical, confirming that it is not a causative factor in altered expression levels (data not shown).



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Figure 2. Expression of atl gene (a) in the related strains sets [lane 1, LLA (GSSA); lane 2, LLE (hGISA); lane 3, PC1 (hGISA); lane 4, PC3 (GISA); lane 5, LIM1 (hGISA); lane 6, LIM3 (GISA); lanes 7–10, GSSA; lane 11, Mu50 (GISA); lane 12, Mu3 (hGISA)], and (b) in all other GISA (lanes 1–6: MI, NJ, GL3700, GL3759, SL, SW307), hGISA (lanes 7–13: FDuf, SH23, AGN, SL6096, NW1018, SW309, SMH2) and GSSA (lanes 14–19). Lane 20, representative 16S control.

 
This study is in accordance with the findings of two previous reports. In the first case, a single laboratory-generated GISA mutant (COL-VR1) showed reduced methicillin-induced autolysis compared with a parental stain, and lower intensity of a >83 kDa band in zymographic profiles, which was thought by the authors to be related to the atl gene product.5 In the second case a clinically related hGISA and GISA pair (IL-A, IL-F) exhibited reduced susceptibility to TIA and similar hydrolase profiles, except for a 116 kDa product, which was less abundant in both isolates compared with a vancomycin-susceptible parent.7

Although it is assumed that the characteristic thickened cell walls in GISA and hGISA may account for lowered susceptibility to TIA, this does not seem to be the case for Mu50 and Mu3, which appear to be exceptions of the phenotype in this respect. This suggests that the thickened cell wall is not primarily responsible for the decreased autolysis susceptibility and it is possible that cell wall composition is different in GISA, or that protease production is increased giving rise to reduced autolysin levels and hence varied autolysis profiles.

In this study all GISA (except Mu50) and six of 11 hGISA showed reduced susceptibility to lysostaphin and TIA, with eight of nine GISA and all hGISA exhibiting lowered expression of atl. The atl sequences of all GISA/hGISA strains studied here were identical to those of two GSSA strains, N315 and MW2,8 which indicates that the altered expression did not result from faulty primer annealing. Exceptions included Mu50, Mu3, NJ and LLA, the former two exhibiting lowered atl expression but similar autolytic susceptibility to GSSA, NJ exhibiting resistance to TIA but not lowered atl expression and the latter, LLA, showing reduced susceptibility to lysostaphin and TIA and expression of atl. As atl plays a fundamental role in cell division and separation, lowered expression may produce build-up of peptidoglycan layers contributing to a thickened cell wall. In NJ, the gene appears to be functioning normally; however, post-translational modification may occur to reduce the activity of Atl, or an increase in protease production may be responsible for diminished lysostaphin and Triton X-100 susceptibility.9 Previously, a two-step hypothesis model was proposed for GISA where lowered susceptibility to TIA precedes the development of vancomycin-intermediate resistance.1 This theory could be expanded to include reduced expression of the atl gene prior to reduced susceptibility to TIA. In the related clinical strains LLA (GSSA) and LLE (hGISA), both atl expression and susceptibility to TIA was reduced in LLA compared with other GSSA, suggesting that autolysis resistance may occur prior to the onset of the hGISA phenotype. These characteristics would contribute to a thicker cell wall, and in turn, decrease the susceptibility of the cell to induced autolysis. This hypothesis has exceptions, such as Mu50, the archetypal GISA in terms of vancomycin susceptibility, but not in terms of either autolytic resistance or sequence comparisons in certain genes.7 This suggests that the GISA/hGISA phenotype, including thickened cell wall, is not mediated through a single set of events, but can be achieved by alternate means.


    Acknowledgements
 
Many thanks to all providers of clinical GISA and hGISA isolates.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1. Boyle-Vavra S, Challapalli M, Daum RS. Resistance to autolysis in vancomycin-selected Staphylococcus aureus isolates precedes vancomycin-intermediate resistance. Antimicrob Agents Chemother 2003; 47: 2036–9.[Abstract/Free Full Text]

2. Koehl JL, Muthaiyan A, Jayaswal RK et al. Cell wall composition and decreased autolytic activity and lysostaphin susceptibility of glycopeptide-intermediate Staphylococcus aureus. Antimicrob Agents Chemother 2004; 48: 3749–57.[Abstract/Free Full Text]

3. Wootton M, Howe RA, Hillman R et al. A modified population analysis profile (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital. J Antimicrob Chemother 2001; 47: 399–403.[Abstract/Free Full Text]

4. Sugai M, Fujiwara T, Ohta K et al. epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus aureus and Staphylococcus capitis. J Bacteriol 1997; 179: 4311–8.

5. Komatsuzawa H, Ohta K, Yamada S et al. Increased glycan chain length distribution and decreased susceptibility to moenomycin in a vancomycin-resistant Staphylococcus aureus mutant. Antimicrob Agents Chemother 2002; 46: 75–81.[Abstract/Free Full Text]

6. Boyle-Vavra S, Labischinski H, Ebert CC et al. A spectrum of changes occurs in peptidoglycan composition of glycopeptide-intermediate-resistant clinical Staphylococcus aureus isolates. Antimicrob Agents Chemother 2001; 45: 280–7.[Abstract/Free Full Text]

7. Boyle-Vavra S, Carey RB, Daum RS. Development of lysostaphin and vancomycin resistance in pre-GISA vancomycin tolerant, methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 2001; 48: 617–25.

8. Wootton M, Avison MB, Howe RA et al. Genetic analysis of seventeen genes in vancomycin intermediate Staphylococcus aureus (VISA) and hetero(h)VISA phenotype. J Antimicrob Chemother 2004; 53: 406–7.[Free Full Text]

9. Shaw LN, Golonka E, Szmyd G et al. Cytoplasmic control of premature activation of a secreted protease zymogen: deletion of staphostatin B (SspC) in Staphylococcus aureus 8325-4 yeilds a profound pleitropic phenotype. J Bacteriol 2005; 187: 1751–62.[Abstract/Free Full Text]