Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis

Reviewer #1 (Public Review):

Summary:
In this work the authors provide evidence that impairment of cell envelope protein homeostasis through blocking the machinery for disulfide bond formation restores the efficacy of antibiotics including beta-lactam drugs and colistin against AMR in Gram-negative bacteria.

Strengths:

The authors employ a thorough approach to showcase the restoration of antibiotic sensitivity through inhibition of the DSB machinery, including the evaluation of various antibiotics on both normal and Dsb-deficient pathogenic bacteria (i.e. Pseudomonas and Stenotrophomonas). The authors corroborate these findings by employing Dsb inhibitors in addition to delta dsbA strains. The methodology is appropriate and includes measuring MICs as well as validating their observations in vivo using the Galleria model.

Weaknesses:

The study would benefit from presenting raw data in some cases, such as MIC values and SDS-PAGE gels, by clarifying the number of independent experiments used, as well as further clarification on statistical significance for some of the data.

Reviewer #2 (Public Review):

Summary:

This work by Kadeřábková et al. demonstrates the importance of a specific protein folding system to effectively folding β-lactamase proteins, which are responsible for resistance to β-lactam antibiotics, and shows that inhibition of this system sensitize multidrug-resistant pathogens to β-lactam treatment. In addition, the authors extend these observations to a two-species co-culture model where β-lactamases provided by one pathogen can protect another, sensitive pathogen from β-lactam treatment. In this model, disrupting the protein folding system also disrupted protection of the sensitive pathogen from antibiotic killing. Overall, the data presented provide a solid foundation for subsequent investigations and development of inhibitors for β-lactamases and other resistance determinants. This and similar strategies may have particular application to polymicrobial contexts, but the present state of knowledge regarding the existence and clinical effects of microbial interactions in disease, both specifically regarding S. maltophilia and P. aeruginosa as well as generally, is significantly overstated.

Strengths:

The authors use clear and reliable molecular biology strategies to show that β-lactamase proteins from P. aeruginosa and Burkholderia species, expressed in E. coli in the absence of the dsbA protein folding system, are variably less capable of resisting the effects of different β-lactam antibiotics compared to the dsbA-competent parent strain (Figure 1). The appropriate control is included in the supplemental materials to demonstrate that this effect is specifically dependent on dsbA, since complementing the mutant with an intact dsbA gene restores antibiotic resistance (Figure S1). The authors subsequently show that this lack of activity can be explained by significantly reduced protein levels and loss-of-function protein misfolding in the dsbA mutant background (Figure 2). These data support the importance of this protein folding mechanism in the activity of multiple clinically relevant β-lactamases.

Native bacterial species are used for subsequent experiments, and the authors provide important context for their antibiotic choices and concentrations by referencing the breakpoints that guide clinical practice. In Figure 4, the authors show that loss of the DsbA system in P. aeruginosa significantly sensitizes clinical isolates expressing different classes of β-lactamases to clinically relevant antibiotics. The appropriate control showing that the dsbA1 mutation does not result in sensitivity to a non-β-lactam antibiotic is included in Figure S2. The authors further show, using an in vivo model for antibiotic treatment, that treatment of a dsbA1 mutant results in moderate and near-complete survival of the infected organisms. The importance of this system in S. maltophilia is then investigated similarly (Figure 5), showing that a dsbA dsbL mutant is also sensitive to β-lactams and colistin, another antibiotic whose resistance mechanism is dependent on the DsbA protein folding system. Importantly, the authors show that a small-molecule inhibitor that disrupts the DsbA system, rather than genetic mutations, is also capable of sensitizing S. maltophilia to these antibiotics. It should be noted that while the sensitization is less pronounced, this molecule has not been optimized for S. maltophilia and would be expected to increase in efficacy once this is done. Together, the data support that interference with the DsbA system in native hosts can sensitize otherwise resistant pathogens to clinically relevant antibiotic therapy.

Finally, the authors investigate the effects of co-culturing S. maltophilia and P. aeruginosa (Figure 5E). These assays are performed in synthetic cystic fibrosis sputum medium (SCFM), which provides a nutritional context similar to that in CF but without the presence of more complex components such as mucin. The authors show that while P. aeruginosa alone is sensitive to the antibiotic, it can survive moderate concentrations in the presence of S. maltophilia and even grow in higher concentrations where S. maltophilia appears to overproduce its β-lactamases. However, this protection is lost in S. maltophilia without the DsbA protein folding system, showing that the protective effect depends on functional production of β-lactamase. The data support a protective role for DsbA-dependent β-lactamase under these co-culture conditions.

Weaknesses:

While Figure 5E demonstrates a protective effect of DsbA-dependent β-lactamase, the omission of CFU data for S. maltophilia makes it difficult to assess the applicability of the polymicrobial strategy. Since S. maltophilia is pre-cultured prior to the addition of P. aeruginosa and antibiotics, it is unclear whether the protective effect is dependent on high S. maltophilia CFU. It is also unclear what the fate of the S. maltophilia dsbA dsbL mutant is under these conditions. If DsbA-deficient S. maltophilia CFU is not impacted, then this treatment will result in the eradication of only one of the pathogens of interest. If the mutant is lost during treatment, then it is not clear whether the loss of protection is due specifically to the production of non-functional β-lactamase or simply the absence of S. maltophilia.

The alleged clinical relevance and immediate, theoretical application of this approach should be properly contextualized. At multiple junctures, the authors state or suggest that interactions between S. maltophilia and P. aeruginosa are known to occur in disease or have known clinical relevance related to treatment failure and disease states. For instance, the citations provided for S. maltophilia protection of P. aeruginosa in the CF lung environment both describe simplified laboratory experiments rather than clinical or in vivo observations. Similarly, the citations provided for both the role of S. maltophilia in treatment failure and CF disease severity do not support either claim. The role of S. maltophilia in CF is currently unsettled, with more recent work reporting conflicting results that support S. maltophilia as a marker, rather than cause, of severe disease. These citations also do not support the suggestion that S. maltophilia specifically contributes to treatment failure. While it is reasonable to pursue these ideas as a hypothesis or potential concern, there is no evidence provided that these specific interactions occur in vivo or that they have clinical relevance.

Reviewer #3 (Public Review):

Summary:
In the face of emerging antibiotic resistance and slow pace of drug discovery, strategies that can enhance the efficacy of existing clinically used antibiotics are highly sought after. In this manuscript, through genetic manipulation of a model bacterium (Escherichia coli) and clinically isolated and antibiotic resistant strains of concern (Pseudomonas, Burkholderia, Stenotrophomonas), an additional drug target to combat resistance and potentiate existing drugs is put forward. These observations were validated in both pure cultures, mixed bacterial cultures and in worm models. The drug target investigated in this study appears to be broadly relevant to the challenge posed by lactamases enzyme that render lactam antibiotics ineffective in the clinic. The compounds that target this enzyme are being developed already, some of which were tested in this study displaying promising results and potential for further optimization by medicinal chemists.

Strengths:
The work is well designed and well executed and targets an urgent area of research with the unprecedented increase in antibiotic resistance.

Weaknesses:
The impact of the work can be strengthened by demonstrating increased efficacy of antibiotics in mice models or wound models for Pseudomonas infections. Worm models are relevant, but still distant from investigations in animal models.