Objectives

To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement.

Methods

We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller–Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates.

Results

Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm.

Conclusions

Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.

中文翻译：

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难治性嗜血杆菌属的甲氧苄氨嘧啶-磺胺甲基异恶唑敏感性测试方法的评估。

目标

为了比较耐甲氧苄啶对磺胺甲恶唑的决定因素和确定的敏感性嗜血嗜血杆菌属菌种的敏感性，为优化甲氧苄氨嘧啶对磺胺甲恶唑的测定提供建议。

方法

我们在Bellvitge大学医院分别收集了50株流感嗜血杆菌和副流感嗜血杆菌菌株。分别使用EuCAST和CLSI标准，使用Mueller-Hinton耐性（MH-F）培养基和嗜血杆菌测试培养基（HTM）分别通过微量稀释，E-test和纸片扩散测试了甲氧苄啶-磺胺甲基恶唑的敏感性。在全基因组测序的分离物中鉴定出folA，folP和其他抗性决定因素的突变。

结果

在HTM上生长时，菌株表现出比MH-F更高的甲氧苄啶-磺胺甲基异恶唑耐药率，与所使用的方法无关（流感嗜血杆菌的平均MIC高2.6倍，副流感嗜血杆菌的平均MIC高1.2倍）。主电阻相关的决定如下：I95L和F154S / V在folA ; folP中的3和15 bp插入和取代；获得sul基因; FolA过度生产可能与-35和-10启动子基序的突变有关。值得注意的是，在19株流感嗜血杆菌中有2株（占10.5％），在33株副流感嗜血杆菌中有9株通过微稀释法被鉴定为具有抗药性的突变菌株（27.3％）被认为不易受到椎间盘扩散的影响。通过将EUCAST指南的临床耐药性折点提高到≤30mm，可以解决这种误解。

结论

考虑到常规使用圆盘扩散法，尽管有与耐药相关的突变，但仍有许多菌株可能被误分类为对甲氧苄啶-磺胺甲基异恶唑敏感。EUCAST MH-F指南给出的当前临床耐药性折点的简单修改可确保正确解释并与微量稀释的参考标准方法相关联。