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
Received 24 May 2005; returned 3 June 2005; revised 25 July 2005; accepted 26 July 2005
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Abstract |
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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
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Introduction |
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Materials and methods |
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Glycopeptide susceptibility was determined by population analysis profilearea under curve (PAP-AUC) using the criteria: <0.9, GSSA; 0.91.29, 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 brainheart 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 KruskalWallis 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 0.0664 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 RTPCR (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).
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Results and discussion |
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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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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.
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Acknowledgements |
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