Malhotra-Kumar Surbhi, Mazzariol Annarita, Van Heirstraeten Liesbet, Lammens Christine, de Rijk Peter, Cornaglia Giuseppe, Goossens Herman
Department of Medical Microbiology, Vaccine and Infectious Disease Institute, Universiteit Antwerpen, Antwerp, Belgium.
J Antimicrob Chemother. 2009 Jan;63(1):42-6. doi: 10.1093/jac/dkn432. Epub 2008 Oct 24.
We identified erm(A)-harbouring Streptococcus pyogenes that expressed three variant phenotypes: (1) low-level resistance to erythromycin (MICs 1-4 mg/L) but high azithromycin MICs in absolute terms (16-64 mg/L; n=6); (2) same as (1) but with a high clindamycin MIC (256 mg/L; n=1); and (3) high-level constitutive MLS (cMLS) resistance (n=1). Here we analysed the genetic basis of these novel phenotypes.
The presence of erm(A) and the absence of macrolide/lincosamide resistance genes erm(B), mef and cfr were confirmed by PCR. erm(A), 23S rRNA, L4 and L22 genes were sequenced. Mutant erm(A) genes were cloned and electrotransformed into the macrolide-susceptible Escherichia coli AG100A. Clonality was determined by emm typing and PFGE. Effects of the identified mutations on free energy changes (DeltaG) and putative configurations of the leader sequence were studied in silico.
Point mutations (G98A, A137C, C140T and G205A) were observed in the erm(A) regulatory region of all eight erm(A)-harbouring S. pyogenes. Five and two isolates belonged to emm77 and emm89 clones, respectively, and one isolate was an emm1. E. coli transformed with mutant erm(A) harbouring G98A, A137C or C140T mutations (phenotypes 1 and 2) did not express high-level azithromycin or clindamycin resistance. However, cMLS resistance was clearly observed in transformants with erm(A) harbouring both A137C and G205A mutations (phenotype 3). In silico analysis showed that DeltaG was minor except for the G205A mutation. Secondary structure predictions further showed that the A137C and G205A mutations together abolished the hairpin sequestering the ribosome-binding and initiation sites of the erm(A) gene, explaining the cMLS phenotype 3.
We report point mutations in the erm(A) regulatory region leading to constitutive methylase expression and the presence of additional, as yet unidentified mechanisms mediating high-level azithromycin and clindamycin resistance in erm(A)-harbouring S. pyogenes.
我们鉴定出携带erm(A)的化脓性链球菌，其表现出三种变异表型：(1)对红霉素低水平耐药（最低抑菌浓度[MIC]为1 - 4mg/L），但阿奇霉素MIC绝对值较高（16 - 64mg/L；n = 6）；(2)与(1)相同，但克林霉素MIC较高（256mg/L；n = 1）；(3)高水平组成型大环内酯类 - 林可酰胺类 - 链阳菌素B（cMLS）耐药（n = 1）。在此，我们分析了这些新表型的遗传基础。
通过聚合酶链反应（PCR）确认erm(A)的存在以及大环内酯类/林可酰胺类耐药基因erm(B)、mef和cfr的缺失。对erm(A)、23S核糖体RNA（rRNA）、L4和L22基因进行测序。将突变的erm(A)基因克隆并电转化到对大环内酯类敏感的大肠杆菌AG100A中。通过emm分型和脉冲场凝胶电泳（PFGE）确定克隆性。在计算机上研究已鉴定突变对前导序列自由能变化（ΔG）和推定构象的影响。
在所有8株携带erm(A)的化脓性链球菌的erm(A)调控区观察到点突变（G98A、A137C、C140T和G205A）。5株和2株分离株分别属于emm77和emm89克隆，1株分离株为emm1型。用携带G98A、A137C或C140T突变（表型1和2）的突变erm(A)转化的大肠杆菌未表现出高水平的阿奇霉素或克林霉素耐药。然而，在携带同时具有A137C和G205A突变的erm(A)的转化子中明显观察到cMLS耐药（表型3）。计算机分析表明，除G205A突变外，ΔG较小。二级结构预测进一步表明，A137C和G205A突变共同消除了使erm(A)基因核糖体结合和起始位点被隔离的发夹结构，解释了cMLS表型3。
我们报告了erm(A)调控区的点突变导致组成型甲基化酶表达，以及在携带erm(A)的化脓性链球菌中介导高水平阿奇霉素和克林霉素耐药的其他尚未明确的机制的存在。
