Revisiting mutational resistance to ampicillin and cefotaxime in Haemophilus influenzae

Abstract

Background: Haemophilus influenzae is an opportunistic bacterial pathogen that can cause severe respiratory tract and invasive infections. The emergence of β-lactamase-negative ampicillin-resistant (BLNAR) strains and unclear correlations between genotypic (i.e., gBLNAR) and phenotypic resistance are challenging empirical treatments and patient management. Thus, we sought to revisit molecular resistance mechanisms and to identify new resistance determinants of H. influenzae.

Methods: We performed a systematic meta-analysis of H. influenzae isolates (n = 291) to quantify the association of phenotypic ampicillin and cefotaxime resistance with previously defined resistance groups, i.e., specific substitution patterns of the penicillin binding protein PBP3, encoded by ftsI. Using phylogenomics and a genome-wide association study (GWAS), we investigated evolutionary trajectories and novel resistance determinants in a public global cohort (n = 555) and a new clinical cohort from three European centers (n = 298), respectively.

Results: Our meta-analysis confirmed that PBP3 group II- and group III-related isolates were significantly associated with phenotypic resistance to ampicillin (p < 0.001), while only group III-related isolates were associated with resistance to cefotaxime (p = 0.02). The vast majority of H. influenzae isolates not classified into a PBP3 resistance group were ampicillin and cefotaxime susceptible. However, particularly group II isolates had low specificities (< 16%) to rule in ampicillin resistance due to clinical breakpoints classifying many of them as phenotypically susceptible. We found indications for positive selection of multiple PBP3 substitutions, which evolved independently and often step-wise in different phylogenetic clades. Beyond ftsI, other possible candidate genes (e.g., oppA, ridA, and ompP2) were moderately associated with ampicillin resistance in the GWAS. The PBP3 substitutions M377I, A502V, N526K, V547I, and N569S were most strongly related to ampicillin resistance and occurred in combination in the most prevalent resistant haplotype H1 in our clinical cohort.

Conclusions: Gradient agar diffusion strips and broth microdilution assays do not consistently classify isolates from PBP3 groups as phenotypically resistant. Consequently, when the minimum inhibitory concentration is close to the clinical breakpoints, and genotypic data is available, PBP3 resistance groups should be prioritized over susceptible phenotypic results for ampicillin. The implications on treatment outcome and bacterial fitness of other extended PBP3 substitution patterns and novel candidate genes need to be determined.

Keywords: Haemophilus influenzae; Ampicillin resistance; BLNAR; Cefotaxime resistance; Genome-wide association study; Haplotype networks; Literature review; MIC; PBP3; Phylogenomics; Resistance groups.

Fig. 1

Distribution of minimum inhibitory concentrations (based on gradient diffusion strips (e.g., Etest)) for A ampicillin (n = 167) and B cefotaxime (n = 127) among previously published β-lactamase negative H. influenzae isolates. The color code represents the main PBP3 groups, isolates with no reported substitutions within the PBP3 transpeptidase domain AA 350–530 (compared to the reference strain Rd KW20), and isolates with miscellaneous PBP3 transpeptidase substitution patterns (not matching the minimum set of mutations used to describe known groups). Hatched bars display isolates exhibiting additional PBP3 substitutions at position 350 to 530, other than the respective group-specific substitutions. Dotted vertical lines indicate clinical breakpoints (EUCAST) that differentiate phenotypically susceptible (S) and phenotypically resistant (R) isolates

Fig. 2

Phylogeny of a global and diverse collection of 555 H. influenzae isolates based on a core-genome alignment of 1429 genes. The category “other” represents all genomes without a PBP3 group-specific substitution or with a PBP3 substitution at position 502 only. Branches that have a local support value of less than 0.8 are highlighted with red dots. AAS amino acid substitutions in PBP3, NTHi non-typeable H. influenzae

Fig. 3

Co-occurrence of PBP3 substitutions in a global H. influenzae collection (n = 555). Hierarchical clustering based on proportions of PBP3 group-specific substitutions on the y-axis that co-occurred with PBP3 substitutions on the x-axis

Fig. 4

The -1 × log10 transformed p-values of variant associations genome-wide. In panels A and B, each dot represents a variant, in panels C and D, each dot represents a gene. A Manhattan plot of MIC GWAS. B Manhattan plot of resistance status GWAS. C Gene-wise most significant MIC-association p-value (x-axis) versus most significant resistance status-associated p-value (y-axis). Whereas C considers all variants occurring in more than 10 isolates, D considers thereof only the subset of variants causing amino acid substitutions. Only ftsI had in both GWASs a much smaller p-value compared to the remaining genes. Respective HTML-based interactive figures providing detailed information on individual data points at mouse-over are provided as Additional file 3: Material S3 (panel A), 4 (panel B), 5, (panel C), and 6 (panel D)

Fig. 5

Haplotype network for 44 ftsI variants detected in a minimum of 10 out of overall 247 isolates. Nodes represent haplotypes, i.e., combinations of variants, and lines connect similar haplotypes. Nodes are scaled according to isolate frequency of the corresponding haplotype and haplotypes are named in the order of occurrence, starting with H1 as the most common haplotype. A Colors denote resistance groups and the abundant combination of group IIb variants with the additional PBP substitution M377I. B Colors denote minimum inhibitory concentrations (MIC) for ampicillin. Blue shades relate to isolates with MIC values considered ampicillin susceptible (MIC < 1 mg/L); red shades indicate isolates classified as ampicillin resistant (MIC > 1 mg/L). Borderline MIC values of 1 mg/L (by definition ampicillin susceptible) are indicated in gray