Molecular Analysis of Rising Fluoroquinolone Resistance in Belgian Non-Invasive Streptococcus pneumoniae Isolates (1995-2014)

We present the results of a longitudinal surveillance study (1995–2014) on fluoroquinolone resistance (FQ-R) among Belgian non-invasive Streptococcus pneumoniae isolates (n = 5,602). For many years, the switch to respiratory fluoroquinolones for the treatment of (a)typical pneumonia had no impact on FQ-R levels. However, since 2011 we observed a significant decrease in susceptibility towards ciprofloxacin, ofloxacin and levofloxacin with peaks of 9.0%, 6.6% and 3.1% resistant isolates, respectively. Resistance to moxifloxacin arised sporadically, and remained gyrA (n = 25), parC (n = 46) and parE (n = 3) in varying combinations, arguing against clonal expansion of FQ-R. The impact of recombination with co-habiting commensal streptococci on FQ-R remains marginal (10.4%). Notably, we observed that a rare combination of DNA Gyrase mutations (GyrA_S81L/GyrB_P454S) suffices for high-level moxifloxacin resistance, contrasting current model. Interestingly, 85/422 pneumococcal strains display MICCIP values which were lowered by at least four dilutions by reserpine, pointing at involvement of efflux pumps in FQ-R. In contrast to susceptible strains, isolates resistant to ciprofloxacin significantly overexpressed the ABC pump PatAB in comparison to reference strain S. pneumoniae ATCC 49619, but this could only be linked to disruptive terminator mutations in a fraction of these. Conversely, no difference in expression of the Major Facilitator PmrA, unaffected by reserpine, was noted between susceptible and resistant S. pneumoniae strains. Finally, we observed that four isolates displayed intermediate to high-level ciprofloxacin resistance without any known molecular resistance mechanism. Focusing future molecular studies on these isolates, which are also commonly found in other studies, might greatly assist in the battle against rising pneumococcal drug resistance.本研究报告了1995年至2014年间针对比利时非侵袭性肺炎链球菌（Streptococcus pneumoniae）分离株（n=5602）开展的氟喹诺酮类耐药性（fluoroquinolone resistance, FQ-R）纵向监测研究结果。多年来，临床改用呼吸喹诺酮类药物治疗（非）典型肺炎的方案，并未对氟喹诺酮类耐药性水平产生影响。但自2011年起，我们观察到肺炎链球菌对环丙沙星、氧氟沙星及左氧氟沙星的敏感性显著下降，三者的耐药分离株占比峰值分别为9.0%、6.6%和3.1%。莫西沙星耐药性则呈零星出现态势，且耐药相关基因gyrA（n=25）、parC（n=46）与parE（n=3）以不同组合形式存在，表明氟喹诺酮类耐药性并非克隆扩增所致。与共生寄居链球菌发生重组对氟喹诺酮类耐药性的影响仍较为有限（仅为10.4%）。值得注意的是，我们发现一种罕见的DNA旋转酶（DNA Gyrase）突变组合（GyrA_S81L/GyrB_P454S）即可导致高水平莫西沙星耐药性，这与现有认知模型相悖。有趣的是，在422株肺炎链球菌中，有85株的环丙沙星最低抑菌浓度（MICCIP）经利血平处理后至少降低了4个稀释梯度，这表明外排泵参与了氟喹诺酮类耐药性的形成。与敏感菌株相比，环丙沙星耐药分离株的ATP结合盒式外排泵（ATP-binding cassette pump, ABC pump）PatAB表达量显著高于参考菌株肺炎链球菌ATCC 49619，但该现象仅在部分耐药株中与终止子突变失活相关。相反，不受利血平影响的主要易化子家族转运蛋白PmrA的表达量，在敏感株与耐药株之间未发现显著差异。最后，我们发现有4株分离株表现出环丙沙星中介至高水平耐药性，但未发现任何已知的分子耐药机制。针对这类在其他研究中也常被报道的分离株开展后续分子研究，或将极大助力于遏制肺炎链球菌耐药性上升的态势。