# A silent sulfonamide-resistant microbial world: The curious case of missing DHPS in candidate phyla radiation

Riyad Razzouk, Adel Azour, Jean-Marc Rolain, Fadi Bittar

► To cite this version:

Riyad Razzouk, Adel Azour, Jean-Marc Rolain, Fadi Bittar. A silent sulfonamide-resistant microbial world: The curious case of missing DHPS in candidate phyla radiation. Journal of Global Antimicrobial Resistance, 2025, 44, pp.241-243. 10.1016/j.jgar.2025.06.024. hal-05386996

HAL Id: hal-05386996

https://hal.science/hal-05386996v1

Submitted on 28 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Short Communication

# A silent sulfonamide-resistant microbial world: The curious case of missing DHPS in candidate phyla radiation

Riyad Razzouk<sup>a,b</sup>, Adel Azour<sup>a,b</sup>, Jean-Marc Rolain<sup>a,c</sup>, Fadi Bittar<sup>a,b,*</sup>

<figure><img src="image_3.png" /></figure>

ARTICLE INFO

Article history:

Received 29 April 2025

Revised 13 June 2025

Accepted 28 June 2025

Available online 8 July 2025

Editor: Xi Li

Keywords:

Candidate phyla radiation

DHFR

DHPS

Sulfamethoxazole

Trimethoprim

A B S T R A C T

Candidate phyla radiation (CPR) microbes exhibit minimal genomes, episymbiotic/parasitic lifestyles, and metabolic dependencies on host cells. In this study, we look for the presence of dihydropteroate synthase (DHPS) and dihydrofolate reductase, key folate biosynthesis enzymes targeted by sulfonamides and trimethoprim, respectively. Using bioinformatic computational methods, analysis of 12 535 (complete and non-complete) CPR genomes revealed the presence of dihydrofolate reductase enzyme in 54% of them, while DHPS was detected in only 6%, suggesting an inherent absence of the folP gene (corresponding to the DHPS). Structural and functional validation confirmed the lack of DHPS activity. These findings indicate an intrinsic resistance to sulfonamides in CPR microbes, raising questions about their adaptation and persistence in antibiotic-rich environments.

© 2025 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

1. Introduction

The candidate phyla radiation (CPR) division has gained significant attention in recent years, with the designation "Candidatus" referring to lineages with low representation and uncultivated microorganisms [1-3]. These microorganisms were primarily identified through metagenomic and single-cell genomic approaches, leading to a revised Tree of Life proposed by Hug et al. [4] and subsequently refined by Castelle et al. [5] and our research team [6]. This phylogenetic framework, based on 16 ribosomal protein markers across diverse taxa, delineates CPR as a distinct microbial clade [4]. Remarkably, CPR organisms constitute over 26% of microbial diversity [4,5] and are found across a wide range of environments, including both natural ecosystems and the human microbiome. Notable representatives include Candidatus Saccharibacteria, prevalent in the human gastrointestinal tract, oral cavity, and vaginal microbiota, as well as Candidatus Peregrinibacteria and the broader Patescibacteria group [7]. Intriguingly, traces of CPR organisms have been detected in the Neanderthal oral microbiome [7], raising fascinating questions about their long-term evolutionary persistence and ecological significance.

CPRs are characterised by their small size, missing synthetic pathways (tricarboxylic acid [TCA]) cycles and electron transport chain), and a compact genome (~1 Mb) [5,6]. They exhibit a cell-surface lifestyle, hence the term "episymbionts", with pili structures attached, mainly to bacterial cells. Additionally, they feature thick layer-like Gram-positive cell walls [5]. Although there are gaps in respiratory pathways, the TCA cycle and gluconeogenesis, the CPR ability to lyse externally acquired elements was hypothesised due to scarce amino-acid breakdown genes [8]. This, in turn, reinforces the hypothesis that they may be parasitic in nature [9]. The absence of *de novo* nucleotide and amino acid synthetic pathways [7] has been documented, suggesting an import from associated host cells (*Schaelia odontolytica* - Saccharibacteria phylum) [10]. For these reasons, we studied the folate biosynthetic pathway, essential to the production of these elements (purines, histidine and methionine amino acids, as well as the dTMP known as deoxythymidine monophosphate). By association, we aimed to study the dihydropteroate synthase (DHPS) enzyme encoded by the *folP* gene, and dihydrofolate reductase (DHFR) enzyme encoded by the *folA* gene within this pathway, as they are targets of sulfonamide-based agents, and trimethoprim drugs, respectively [11].

2. Materials, methods and results

Our study began by acquiring 12 535 CPR genomes available from the NCBI dataset (up to 30 June, 2024) (Table 1) [12] with two

ARTICLE INFO

<footnote><sup>a</sup>Aix Marseille Université, MEPHI, Marseille, France</footnote>

<footnote><sup>b</sup>IHU Méditerranée Infection, Marseille, France</footnote>

<footnote><sup>c</sup>Aix Marseille Université, APHM, MEPHI, Marseille, France</footnote>

<footnote>* Corresponding author. Aix Marseille Université, MEPHI, 19-21 Boulevard Jean Moulin, Marseille, 13005, France

E-mail address: fadi.bittar@univ-amu.fr (F. Bittar).</footnote>
<caption>**Table 1**</caption>

Analysis of the studied CPR phyla complete/non-complete genomes, showing the absence or presence of DHFR and DHPS (trimethoprim-sulfamethoxazole targets, respectively).

<table><thead><tr><th>CPR Classification</th><th>Number of genomes studied</th><th>Number of DHPS sequences per CPR phylum</th><th>Number of DHFR sequences per CPR phylum</th></tr></thead><tbody><tr><td>Candidate division Kazan</td><td>10 (1)</td><td>0 (0)</td><td>2 (0)</td></tr><tr><td>Candidate division WWE3</td><td>173 (1)</td><td>1 (0)</td><td>19 (0)</td></tr><tr><td>Candidatus Absconditabacteria</td><td>55 (3)</td><td>0 (0)</td><td>16 (4)</td></tr><tr><td>Candidatus Berkelbacteria</td><td>108 (1)</td><td>0 (0)</td><td>44 (0)</td></tr><tr><td>Candidatus Dojkabacteria</td><td>169 (2)</td><td>0 (0)</td><td>38 (0)</td></tr><tr><td>Candidatus Doudnabacteria</td><td>86 (0)</td><td>0 (0)</td><td>69 (0)</td></tr><tr><td>Candidatus Gracilibacteria</td><td>278 (2)</td><td>50 (0)</td><td>117 (2)</td></tr><tr><td>Parcubacteria group</td><td>4212 (22)</td><td>100 (0)</td><td>2263 (13)</td></tr><tr><td>Candidatus Peregrinibacteria</td><td>378 (11)</td><td>241 (4)</td><td>282 (12)</td></tr><tr><td>Candidatus Saccharibacteria</td><td>2611 (47)</td><td>150 (1)</td><td>1711 (43)</td></tr><tr><td>Candidatus Wirthbacteria</td><td>3 (0)</td><td>0 (0)</td><td>1 (0)</td></tr><tr><td>Microgenomates group</td><td>2402 (10)</td><td>80 (0)</td><td>1267 (5)</td></tr><tr><td>Unclassified</td><td>1949 (1)</td><td>112 (0)</td><td>838 (1)</td></tr><tr><td>Patescibacteria group</td><td></td><td></td><td></td></tr><tr><td>Total:</td><td>12 434 (101)</td><td>734 (5)</td><td>6667 (80)</td></tr></tbody></table>

The numbers in parentheses represent those concerning the complete genomes or the numbers of DHPS/DHFR sequences found in complete genomes.

complete referential genomes, for *Schaalia odontolytica* host (Gen-Bank accession GCA_009730335.1) and gram-positive bacterium *Staphylococcus aureus* (GenBank accession GCA_001019055.1). Both genomes provided the DHFR and DHPS reference proteins for our alignments. The primary focus was on their alignments against the 101 complete CPR genomes through an in house BLASTp with an e-value threshold of 0.0001, ensuring reliable and conclusive outcomes. This approach resulted in 31 potential DHPS and 90 potential DHFR CPR sequences (Fig. 1). We thereby refined our hits through the NCBI Conserved Domain Database (CDD) tool [13], yielding the BLAST results with the functional domains of each active cluster (cluster results not shown). Those were then compared to the reference bacterial protein domains (i.e., from *S. odontolytica* and *S. aureus*), showing the presence of necessary functioning domains. The elected products were five functional CPR DHPS sequences, and 80 DHFR sequences (shown in Table 1 and Fig. 1). For further proof, we verified the validity and functionality of the resulting CPR sequences from this step, using a supplementary bioinformatics tool, Phyre2 (Protein Homology/AnalogY Recognition Engine) [14]), allowing us to predict possible 3D structures for each protein. Repetitive high rates of confidence (100%) for the generated 3D structures of DHFR or DHPS sequences were observed. Ultimately, we revealed the presence of five DHPS genes (5%) in five CPR genomes and 80 DHFR genes (79%) in 79 CPR genomes (Fig. 1).

Furthermore, we ran another in-house BLASTp for the 12 434 annotated and concatenated non-complete CPR genomes with an e-value of 0.0001 using the verified CPR proteins from the complete genomes, as subjects (Fig. 1). As a result, we identified 3297 and 21 130 additional potential DHPS and DHFR sequences (BLASTp results), respectively. Another CDD search was then conducted on these hits, revealing 734 DHPS and 6667 DHFR annotated proteins with functional domains (Table 1 and Fig. 1). These results are congruent with those of the complete genome analyses with

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Methodology and main results of the DHPS- DHFR isolation, functional verification, and global potential detection using the BLAST tool.</figcaption></figure>

a 6% prevalence for DHPS and a 54% prevalence for the DHFR. Our methodology and main results are presented in more detail in Fig. 1. Interestingly, encoded sequences for DHPS and DHFR were mapped and located randomly within the analysed complete genomes (data not shown).

### 3. Discussion

As CPR cells lack pathways for nucleotide and amino acids, which are essential for survival, it was important to understand the reason behind this claim. Studying the folate biosynthesis pathway within the CPRs, we noticed a relative abundance of the trimethoprim target (DHFR) and a scarcity of the sulfonamide target (DHPS), lending our conclusion a solid foundation. Could this absence be seen as an intrinsic form of resistance? Based on our in-silico analysis of the CPR genomes, we did not detect any
homologs of the DHPS-encoding gene, suggesting that these mi-
crobes may intrinsically lack this gene and could potentially ex-
hibit resistance to sulfonamides. However, this interpretation re-
mains hypothetical and requires experimental validation. Interest-
ingly, a recent study [15] evaluated the spread of microbes, in-
cluding CPR cells, within collected groundwater samples in rela-
tion to the presence of antibiotics. CPRs were relatively more abun-
dant in samples polluted by sulfonamides. Our work suggests that
this could be explained by the absence of the DHPS gene in CPR
microbes. This phenomenon of an incomplete folate pathway can
also be observed in other microbes. One such example is the ab-
sence of the DHFR gene in *Tropheryma whittlei* organism from its
genome, according to in vitro studies [16], rendering its inhibition
by the trimethoprim agent ineffective. Another example of antimi-
crobial resistance and target lack is *Mycoplasma pneumoniae*, which
is intrinsically resistant to beta-lactams due to the lack of cell
walls [17].

Moreover, in contrast to DHPS, the DHFR-like enzyme is rel-
atively abundant within the CPRs. Natural compounds (tea com-
pound, epigallocatechin-gallate (EGCG)) act as antifolates targeting
and inhibiting the DHFR enzyme [18,19]. In turn, is it possible that
CPR DHFR sequences have additional functions? A similar possibil-
ity was previously considered and elaborated upon regarding CPR
beta lactamase proteins not only hydrolysing beta-lactams, but be-
ing additionally equipped with RNA-lytic abilities [20]. The concept
of multiple functionalities was further explored in 2023, when CPR
beta-lactamases were referred to as “metallo-hydrolases” for their
higher lysing capacities [21].

In conclusion, our massive genomic analysis demonstrates that
CPR microbes largely lack the DHPS gene in the folate biosynthetic
pathway, confirming their deficiency in nucleotide and amino acid
biosynthesis and suggesting their intrinsic resistance to sulfon-
amides. Additional experiments are needed to understand the
function of DHFR-like genes in CPR microbes.

Funding

This work was supported by a grant from the French Govern-
ment managed by the National Research Agency under the "In-
vestissements d'avenir (Investments for the Future)" programme
with the reference ANR-10-IAHU-03 (Méditerranée Infection), by
the Contrat Plan Etat-Région and the European funding FEDER IHU-
PERF.

Author contributions

Jean-Marc Rolain and Fadi Bittar: designed the study and drafted and revised the manuscript. **Riyad Razzouk and Adel Azour:** performed bioinformatics analyses. All authors have read and approved the final manuscript.

Declaration of competing interest

None declared.

References

[1] Marcy Y, Ouverney C, Bik EM, Lösekann T, Ivanova N, Martin HG, et al. Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncollectivated TM7 microbes from the human mouth. PNAS 2007;104(29):11889-94. doi:10.1073/pnas.0704662104.

[2] Murugkar PP, Collins AJ, Chen T, Dewhirst FE. Isolation and cultivation of candidate phyla radiation saccharibacteria (TM7) bacteria in coculture with bacterial hosts. J Oral Microbiol 2020;12(1):1814666. doi:10.1080/20002297.2020.1814666.

[3] Brown CT, Hug LA, Thomas BC, Sharon I, Castelle CJ, Singh, et al. Unusual biology across a group comprising more than 15% of domain bacteria. Nat 2015;523(7559):208–11. doi:10.1038/nature14486.

[4] Hug LA, Baker BJ, Anantharaman K, Brown CT, Probst AJ, Castelle CJ, et al. A new view of the tree of life. Nat Microbiol 2016;1:16048. doi:10.1038/nnmicrobiol.2016.48.

[5] Castelle CJ, Banfield JF. Major new microbial groups expand diversity and alter our understanding of the Tree of Life. Cell 2018;172(6):1181-97. doi:10.1016/j. cell.2018.02.016.

[6] Ibrahim A, Colson P, Merhej V, Zgheib R, Maatouk M, Naud S, et al. Rhibosomal reclassification of living organisms. Int J Mol Sci 2021;22(11):5643. doi:10.3390/ijms22115643.

[7] Naud S, Ibrahim A, Valles C, Maatouk M, Bittar F, Tidjani Alou M, et al. (2022). Candidate phyla radiation, an underappreciated division of the Human microbiome, and its impact on health and disease. Clin Microbiol Rev 2022;35(3):e0014021. doi:10.1128/cmr.00140-21.

[8] Anantharaman K, Brown CT, Burstein D, Castelle CJ, Probst AJ, Thomas BC, et al. Analysis of five complete genome sequences for members of the class peribacteria in the recently recognized peregrinibacteria bacterial phylum. PeerJ 2016;4:e1607. doi:10.7717/peerj.1607.

[9] Bernard C, Lannes R, Li Y, Bapteste É, Lopez P. Rich repertoire of Quorum sensing protein coding sequences in CPR and DPANN associated with interspecies and interkingdom communication. mSystems 2020;5(5) e00414-20. doi:10.1128/mSystems.00414-20.

[10] Maatouk M, Ibrahim A, Rolain JM, Merhej V, Bittar F. Small and equipped: the rich repertoire of antibiotic resistance genes in Candidate Phyla radiation genomes. mSystems 2021;6(6):e0089821. doi:10.1128/mSystems.00898-21.

[11] Estrada A, Wright DL, Anderson AC. Antibacterial antifolates: from development through resistance to the next generation. CSH Perspect Med 2016;6(8):1-10. doi:10.1101/cshperspect.a028324.

[12] Ncbi dataset- source available online: https://www.ncbi.nlm.nih.gov/datasets/.

[13] Conserved domain database and resources. https://www.ncbi.nlm.nih.gov/
Structure/bwrpsb/bwrpsb.cgi.

[14] Protein homology/analogY recognition Engine V 2.0 (Phyre2). http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index.

[15] Tang M, Chen Q, Zhong H, Liu S, Sun W. CPR bacteria and DPANN archaea play pivotal roles in response of microbial community to antibiotic stress in groundwater. Water res; 2024;251:121137.

[16] Fenollar F, Rolain JM, Alric L, Papo T, Chauveheid MP, van de Beek D, et al. Resistance to trimethoprim/sulfamethoxazole and tropheryma whittlei. Int J Antimicrob Agents 2009;34(3):255-9. doi:10.1016/j.ijantimicag.2009.02.014.

[17] Pereyre S, Goret J, Bébéar C. Mycoplasma pneumoniae: current knowledge on macrolide resistance and treatment. Front Microbiol 2016;7:974. doi:10.3389/fmicb.2016.00974.

[18] Spina M, Cuccioloni M, Mozzicafreddo M, Montecchia F, Pucciarelli S, Eleu-
teri AM, et al. Mechanism of inhibition of wt-dihydrofolate reductase from E.
coli by tea epigallocatechin-gallate. Proteins 2008;72(1):240-51. doi:10.1002/
prot.21914.

[19] Sánchez-del-Campo L, Sáez-Ayala M, Chazarra S, Cabezas-Herrera J, Rodríguez-López JN. Binding of natural and synthetic polyphenols to Human dihydrofolate reductase. Int J Mol Sci 2009;10(12):5398-410. doi:10.3390/ijms10125398.

[20] Maatouk M, Ibrahim A, Pinault L, Armstrong N, Azza S, Rolain JM, et al. New beta-lactamases in candidate phyla radiation: owning pleiotropic enzymes is a smart paradigm for microorganisms with a reduced genome. Int J Mol Sci 2022;23(10):5446. doi:10.3390/ijms23105446.

[21] Maatouk M, Merhej V, Pontarotti P, Ibrahim A, Rolain JM, Bittar F. Metallo-beta-lactamase-like encoding genes in Candidate Phyla radiation: widespread and highly divergent proteins with potential multifunctionality. Microorganisms 2023;11(8):1933. doi:10.3390/microorganisms11081933.