1 Department of Pathology, Hershey Medical Center, Mail code H083, 500 University Dr., Hershey, PA 17033; 2 Functional Genomic Core Hershey Medical Center, Hershey, PA 17033, USA
Received 21 January 2004; returned 1 April 2004; revised 1 June 2004; accepted 11 June 2004
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Abstract |
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Objective: Determination of macrolide resistance mechanism(s) in hypersusceptible and hyperresistant strains.
Methods: The presence of macrolide efflux in the strains was studied by radioactive erythromycin accumulation. Ribosomal mutations were investigated by sequencing. The possible role of acrAB clusters in macrolide resistance was studied by sequencing and expression analysis.
Results: The parent strain had no ribosomal alteration, but both high-level resistant and hypersusceptible strains had R88P mutations in ribosomal protein L22. Radioactive macrolide accumulation studies pointed to the presence of macrolide efflux in the high-level resistant and parent strains, but not in the hypersusceptible derivative. Transformation of hypersusceptible strains using total DNA from the parent strain restored the macrolide efflux system in the hypersusceptible strain, which was confirmed by MIC levels and radioactive erythromycin accumulation similar to that of the mutant resistant strain. Analysis of sequence and transcription of acrAB gene clusters showed no significant differences between resistant and hypersusceptible derivatives.
Conclusion: Mutation in ribosomal protein L22 alone does not confer high-level macrolide resistance unless efflux is present.
Keywords: macrolide resistance , H. influenzae , macrolide efflux , ribosomal mutations
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Introduction |
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In a previously published study, a strain of H. influenzae, HMC-S, reverted spontaneously from a high-level resistant strain and became hypersusceptible to macrolides.3 The purpose of this study was to identify the mechanism(s) of hypersusceptibility in H. influenzae HMC-S.
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Materials and methods |
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Parent (HMC-P), clarithromycin-selected resistant (HMC-C) and spontaneously reverted hypersusceptible (HMC-S) H. influenzae strains from Clark et al.3 were used in this study. H. influenzae strain Rd was used for transformation experiments. CCCP (carbonyl cyanide m-chlorophenylhydrazone), a protonophore, was purchased from Sigma (Sigma Inc., St Louis, MO, USA). Radioactive erythromycin ([N-methyl-14C]erythromycin 47 Ci/mmol, 2.14 mg/mL) was purchased from Perkin-Elmer (Perkin-Elmer Life Sciences Inc., Boston, MA, USA).
MIC methodology and antimicrobials
Microdilution MICs of all strains were performed, as recommended by the NCCLS, using freshly prepared Haemophilus test medium (HTM), and inoculum checks were carried out in each case.6 Standard quality control strains were included in each run. Cultures were incubated for 1620 h in ambient air. Drugs were obtained from their respective manufacturers.
Erythromycin accumulation
Efflux of erythromycin was determined indirectly by measuring the accumulation of radioactive [N-methyl-14C]erythromycin. Accumulation was conducted as described previously in the presence or absence of 25 mg/L CCCP.2
Determination of macrolide resistance mechanism
The presence of known resistance genes for macrolides mef(A), erm(B), erm(A) [subclass erm(TR)] and ere(A) was tested by PCR as described previously.3 Alterations in the genes coding for 23S rRNA, ribosomal proteins L4 and L22 were verified by sequencing after amplification by PCR as described previously.3
Southern-blot hybridization
The acrA gene was amplified by PCR from the parent strain and labelled by random priming using a Random Prime Labelling System (Amersham Biosciences, Buckinghamshire, UK). Chromosomal DNA of parent, hypersusceptible, high-level resistant and transformed strains was digested with EcoRI and HindIII, separated on a 1% (w/v) agarose gel and transferred to a HybondN+ nylon membrane (Amersham Pharmacia Biotech, Uppsala, Sweden) under alkaline conditions using a vacuum blotter (Bio-Rad, USA). Hybridization was carried out under highly stringent conditions using the ECL detection system and protocol (Amersham Biosciences, Buckinghamshire, UK).
Transformation
Total DNA was extracted from H. influenzae HMC-P strain and used for transformation of hypersusceptible strain HMC-S, as described previously by Barcak et al.7 Transformants were selected on brain heart infusion agar (Becton Dickinson, Cockeysville, MD, USA) supplemented with haematin (15 mg/L) and NAD (15 mg/L) (sBHI), that contained erythromycin 4 mg/L.
Sequencing
Genes were amplified using specific primers and sequenced (Table 1). PCR products, after amplification of acrA and acrB genes, were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). Nucleotide sequences of the amplified genes were obtained by direct sequencing using the CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA). acrAB clusters were compared with those from the complete genome sequence of H. influenzae Rd KW20, using ClustalW software (www.ebi.ac.uk/clustalw/). Primers used for PCR amplification and sequencing are listed in Table 1.
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RNA was prepared using an RNeasy kit with an RNA protection bacteria reagent (Qiagen, Valencia, CA, USA) as recommended by the manufacturer.
Quantitative real-time PCR
Oligonucleotide primers and probes for acrA and 16S rRNA were designed and purchased from IDT Inc. (Evanston, IL, USA). Probes consisted of an oligonucleotide labelled at the 5' end with the reporter dye 6-carboxyfluorescein (FAM) and with the quencher dye N,N',N'-tetramethyl-6 carboxytetramethylrhodamine (TAMRA) at the 3' end. QRTPCR was performed using the QuantiTect Probe PCR kit, as described by the manufacturer (Qiagen, Valencia, CA, USA).
The quantity of total RNA was assessed using an Aligent 2100 Bioanalyser, which confirmed that the total RNA was not contaminated and also that there was no presence of contaminating DNA. Reverse transcription was carried out using SuperScript III (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer.
Amplification and detection of specific products were performed with the ABI Prism 7700 sequence detection system (PE Applied Biosystems). The QRTPCR thermal cycling conditions were as follows: HotStarTaq DNA polymerase activation step of 95°C for 15 min, followed by 40 cycles of PCR consisting of a denaturation step of 94°C for 15 s and a combined annealing/extension step of 60°C for 1 min. The critical threshold cycle (Ct) was defined as the cycle at which the fluorescence became detectable above background levels and was inversely proportional to the logarithm of the initial number of template molecules. The relative quantities of each amplicon were calculated from these Ct values using the relative standard curve method of quantification, according to the manufacturer's instructions (Applied Biosystems). The quantity of cDNA for each experimental gene was normalized to the quantity of 16S cDNA in each sample. Each RNA sample was run in triplicate and repeated twice. The primer sets 16SHF128162F 5'-ATCGGAATAACTGGGCGTAA and 16SHF128057R 5'-GTACTCTAGTTACCCAGTCT, and ACRA2167F2 5'-ATTGCCTATTTACTTGAACC and ACRA2282R1 5'-TGACGATCTTTAGTAAATACG were used to amplify a 106 bp and 115 bp fragment of the 16S rRNA and acrA genes, respectively. Probes, labelled with FAM at the 5' end and TAMRA at the 3' end, were acrAPACRA2227 5'-AAATTGGATCGTCTCTATCGTGCG and 16SHF128100 5'-AAGTGAGGTGTGAAAGCCCTGG. Specificity of the PCR was verified by ethidium bromide staining after electrophoresis on 3.5% agarose gels.
Microarray transcription analysis
The microarray procedure was as previously described by J. L. DeRisi at http://www.microarrays.org/pdfs/amino-allyl-protocol.pdf, with additions and/or modifications outlined below. Oligonucleotide probes (70-mer) representing all ORFs present in the H. influenzae RD strain were purchased from Qiagen Operon (Alameda, CA, USA) and printed onto epoxysilane slides from MWG Biotech (High Point, NC, USA) by means of a Biorobotic Microgrid II array spotter from Genomic Solutions (Ann Arbor, MI, USA). Total RNA was extracted from H. influenzae strains grown to mid-log phase. RNA was used for indirect labelling with AlexaFluor dyes from Molecular Probes, Inc. (Eugene, OR, USA). The data were analysed using GeneSpring software (Silicon Genetics, Redwood City, CA, USA).
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Results |
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All three strains were tested by PCR for the presence of erm and mef genes, and by PCR and subsequent sequencing for mutations in genes for ribosomal proteins L4 and L22, and 23S rRNA. No erm or mef genes were detected. The parent strain had no ribosomal alterations, whereas resistant and hypersusceptible strains had mutations in ribosomal protein L22 by substitution of arginine by proline at position 88 (Table 2).
Total DNA was extracted from H. influenzae HMC-P and used for transformation of H. influenzae HMC-S. Transformant HMC-SHMC-P became more resistant to macrolides than the parent strain, which showed the importance of the L22 mutation to the contribution of macrolide resistance. The erythromycin MICs were 8, 0.5 and 64 mg/L for parent, hypersusceptible and transformant strains, respectively. The activity of some dyes and detergents, usually substrates for multidrug efflux pumps, were tested. MICs for acriflavin, ethidium bromide and SDS were low in the hypersusceptible strain compared with parent and transformant strains. HMC-P and the transformant had four- and two-, eight- and 16-, and four-fold higher MICs than HMC-S for acriflavin, ethidium bromide and SDS, respectively. The parent strain had a four-fold higher rifampicin MIC than hypersusceptible and hyperresistant derivatives (Table 2).
Accumulation of macrolides
The mechanism of H. influenzae HMC-S hypersusceptibility was investigated by measuring the accumulation of radioactive erythromycin in the presence and absence of CCCP. Results are shown in Figure 1. The accumulation of radioactive erythromycin was low for HMC-P, but increased after CCCP treatment. This difference may indicate the presence of a macrolide efflux mechanism in this strain. Accumulation was already high for the hypersusceptible strain, and CCCP treatment did not affect the level of accumulation in HMC-S, suggesting the loss of an efflux mechanism in this strain.
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The location and the presence of the acrAB gene clusters were studied by Southern blot in parent, hyperresistant, hypersusceptible and transformant strains. All strains had the acrA gene on the same size fragments; a 12 000 bp EcoRI fragment and a 16 000 bp HindIII (data not shown). The difference in expression of the acrAB gene cluster between parent, hyperresistant and hypersusceptible strains was investigated.
The expression of acrAB cluster was quantified by real-time RTPCR. The acrA mRNA was measured in the hypersusceptible strain and compared with parent and hyperresistant derivatives. Quantitive real-time RTPCR analysis showed that the expression levels of the acrA gene in hyperresistant and hypersusceptible derivatives were similar; the amount of expression of acrA mRNA/16S rRNA was 0.93 for HMC-S, whereas it was 1.08 for HMC-P and 1.01 for HMC-C.
Sequence analysis
The acrA and acrB genes from HMC-P and HMC-S showed >90% homology with the sequence of the H. influenzae Rd strain. Two amino acid changes were observed in the AcrB protein by P660A and Q825H substitution when compared with AcrB from the H. influenzae Rd strain. However, both HMC-P and HMC-S had the same amino acid sequence. The deduced amino acid sequence of AcrA protein showed differences of 12 amino acids: M36I, G48E, M83L, A91T, V92I, V163L, S165N, A172V, E242K, I360V, V361A and K365X. The only amino acid change between the susceptible and parent strain was in AcrAa V357G substitution.
Microarray transcription profiling
Analysis of transcriptomes from hyperresistant and hypersusceptible strains showed that there were no differences in expression between acrA and acrB genes. The normalized fold changes for HMC-C versus HMC-S and HMC-SHMC-P versus HMC-S were 1.36±0.37 and 1.17±0.31 for acrB and 0.75±0.36 and 0.72±0.45 for acrA, respectively. The expression of genes in HMC-S was compared with the expression in HMC-C and the transformant HMC-S
HMC-P. High-level expression of 28 genes was detected in both hyperresistant strains. Two of these genes are ABC transporters (one peptide ABC transporter the other an arginine ABC transporter) (Figure 2).
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Discussion |
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Even though we could not detect any differences in acrAB cluster expression of hypersusceptible and mutant resistant strains, the macrolide accumulation test has indicated the presence of a macrolide efflux mechanism in parent and mutant resistant strains absent in hypersusceptible strains. The effect of alterations in the L22 protein on macrolide susceptibility have been shown in Staphylococccus aureus,9 Streptococcus pneumoniae10 and in H. influenzae.2 Transformation of a susceptible S. pneumoniae and S. aureus with an altered L22 gene causes increased macrolide MICs in these strains.9,10 The H. influenzae Rd strain has been shown to become macrolide resistant after acquiring an R88P change in L22 by transformation.3 The L22 protein is important for assembly of the 50S ribosomal subunit and for the folding of 23S rRNA.11 The positively charged arginine 88 is located at the tip of the ß hairpin and interacts with the negatively charged phosphate group of the RNA (Figure 3).9,12 The substitution of positively charged arginine in ribosomal protein L22 might affect the 23S rRNA folding, cause conformational change in the ribosome and alter erythromycin activity. Even though R88P mutation did not affect the erythromycin activity in hypersusceptible strain HMC-S in the absence of efflux, restoration of the erythromycin efflux mechanism caused high-level macrolide resistance by association of two resistance mechanisms, efflux and ribosomal mutation. The importance of the association of the efflux mechanism with target alterations has been reported for quinolone resistance, for which the association of topoisomerase alterations and the presence of an efflux pump (AcrAB) is necessary.13 However, results from the present study indicate the possible presence of an efflux mechanism other than the acrAB cluster, because no significant differences were found in the expression of the acrAB cluster. Only one amino acid change (V357G) was observed in the sequence of the AcrA protein. The role of other efflux pumps, including the up-regulated ABC transporters, in macrolide efflux is not known yet and is under investigation.
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Acknowledgements |
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Footnotes |
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Present address. PLIVAResearch Institute Ltd., Prilaz baruna Filipovica 29, 10000 Zagreb, Croatia
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References |
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