Haemophilus parainfluenzae is an opportunistic Gram-negative pathogen that colonizes the respiratory and urogenital tracts (Nørskov-Lauritsen, 2014). This species has been implicated as a cause for various diseases, including respiratory tract infections (Kosikowska et al., 2016), endocarditis (Revest et al., 2016) and arthritis (Baron et al., 2020), with a notably high prevalence as a cause of urethritis in recent years (Ducours et al., 2020; Hsu et al., 2015; Magdaleno-Tapial et al., 2019).

Polysaccharide capsules, a major virulence factor in H. influenzae, have also been identified in H. parainfluenzae, although their precise role in this species remains unclear. In H. influenzae, these capsules are crucial for evading host immune responses and have been used in vaccine development (González-Díaz et al., 2019; Sierra et al., 2021; Watts and Holta, 2019). The genetic blueprint for these polysaccharide capsules in the genus Haemophilus is encoded in the cap locus, which comprises three different regions. Region I has four conserved genes (bexABCD) that encode the export apparatus for the translocation of capsular polysaccharides. Region II contains serotype-specific genes (three to eight genes) involved in the polysaccharide biosynthesis. Finally, region III has two genes (hcsAB) responsible for polysaccharide transport across the outer membrane (Potts et al., 2019). To date, three different capsular operons have been described in H. parainfluenzae: HPAR-type1 (González-Díaz et al., 2019) and HPAR-type2 (Sierra et al., 2021), both detected in urogenital strains, and the serotype f-like operon recently identified in colonizing strains from nasopharyngeal samples of healthy children in Portugal (Bajanca-Lavado et al., 2022).

Along with virulence factors, antimicrobial resistance has emerged as a significant concern in H. parainfluenzae. The increasing incidence of multidrug resistance in this species is attributed to target-gene mutations and the acquisition of transferable resistance genes. Extensively drug-resistant (XDR) strains have been detected in recent years, particularly in urogenital samples, showing resistance to β-lactams, fluoroquinolones, macrolides, chloramphenicol, trimethoprim-sulfamethoxazole and tetracycline. Notably, our group previously reported the presence of a CTX-M-15 extended spectrum β-lactamase (ESBL) in four XDR H. parainfluenzae strains isolated from three urethral exudates and a genital ulcer (Saiz-Escobedo et al., 2023). This finding was subsequently corroborated by the identification of the CTX-M-15 ESBL in a urogenital XDR H. parainfluenzae strain from France (Caméléna et al., 2024).

The main objective of this study is to characterize a novel capsular operon detected in two XDR H. parainfluenzae strains isolated from genital ulcers in MSM patients. Additionally, we also aim to provide a comprehensive genotypic and phenotypic characterization of these capsulated strains, with particular emphasis on their antimicrobial resistance profiles.

The identification of a novel capsular operon, HPAR_type4, in two H. parainfluenzae strains isolated from genital ulcers in MSM patients expands our understanding of the genetic diversity of this species. This capsular operon was identified in two H. parainfluenzae strains isolated from different hospitals, with the episodes occurring 2 years apart. While there is no evidence suggesting direct contact between the patients, which initially appears to exclude a direct epidemiological link, the genetic similarity between the strains, sharing 96.7% homology, raises questions about potential common sources or transmission pathways. The absence of a standardized clonal classification system for H. parainfluenzae complicates the assessment of the epidemiological relationship between these strains. Further investigation is needed to determine if these cases represent an emerging trend or are isolated events.

Continuous genomic monitoring led to the detection of the first capsular operon in H. parainfluenzae (González-Díaz et al., 2019). Five years after its publication, four different capsular operons have already been found in this species (González-Díaz et al., 2019; Sierra et al., 2021; Bajanca-Lavado et al., 2022), including the HPAR_type4 operon described in this study. The presence of capsules in this genus may enhance their ability to evade host immune responses, as demonstrated in H. influenzae, where capsulated strains often exhibit increased pathogenicity (Guellil et al., 2022). Although the capsular operons found in H. parainfluenzae differ structurally from the virulent H. influenzae type b capsule, the notable capacity of this species to acquire new genetic material raises concerns about the potential emergence of similarly virulent capsules in the future (Sierra et al., 2021). Thus, its surveillance is important to monitor and respond to any emerging virulence factors.

The genetic organization of the capsular operon identified in HUB-HP17268 and HUMT-HP05 aligns with other Haemophilus spp. capsular loci, comprising three regions. The high homology of regions I and III with the previously characterized H. parainfluenzae HPAR_type1 operon (González-Díaz et al., 2019), indicates a conserved mechanism for capsular polysaccharide biosynthesis and export within the genus. The serotype-specific region II contains ORFs similar to genes from both H. parainfluenzae HPAR_type1 (pcsA) and H. sputorum HSPU_type1 (cpsB and cpsC), suggesting that horizontal gene transfer and recombination may play a role in the evolution of capsular diversity among Haemophilus species (Nielsen et al., 2015). Both strains were isolated from distinct Catalan towns over a 2-year period (without epidemiological connections), which excludes definitive conclusions about clonal transmission. However, the 17 recombinant blocks and the acquisition of CTX-M-15-bearing ICEHpaHUB6 in HUMT-HP05 demonstrate the capacity of H. parainfluenzae for genomic recombination.

Since the first report of a capsular H. parainfluenzae strain, we have observed an alarming increase in antibiotic resistance. Both strains in our study exhibited XDR profiles, including resistance to β-lactams and other broad-spectrum antibiotics. This trend is consistent with our previous observations of capsulated MDR and XDR strains (Sierra et al., 2021). Interestingly, the first XDR strain described in the literature also carried an HPAR_type1 operon. We were able to identify this operon through genomic analysis of the sequence deposited in NCBI, as this detail was not reported in the original article (Tinguely et al., 2013). Since then, ESBL production has also been identified in this species. Initially, the bla_CTX-M-15 gene was detected in four strains at our hospital (Saiz-Escobedo et al., 2023). Subsequently, another strain (ASM3628892v1) carrying the same resistance determinant was identified in France, with the particularity that this strain also carried the H. parainfluenzae HPAR_type1 operon (Caméléna et al., 2024). The identification of the CTX-M-15 ESBL in HUMT-HP05 highlights the ability of H. parainfluenzae to acquire resistance genes through mobile genetic elements (Juhas et al., 2007). The insertion of bla_CTX-M-15 into the ICEHpaHUB6 element may facilitate the spread of this resistance determinant within the species and among other bacteria co-colonizing the urogenital tract. Its detection in an MSM patient further underscores the importance of considering this pathogen in the context of STI management, especially in cases of co-infection with other pathogens, as it may serve as a reservoir for resistance genes.

To date, only transposons and ICE structures had been detected integrated into the H. parainfluenzae genome (Sierra et al., 2021). This study identified two previously unreported plasmids, both carrying genes essential for plasmid maintenance, replication and segregation, indicating their potential for propagation within host cells (Smillie et al., 2010). Among them, we identified a plasmid in H. parainfluenzae with significant homology to a plasmid from A. actinomycetemcomitans, a member of the same Pasteurellaceae family. This finding highlights the potential for horizontal transfer within this taxonomic group, which could contribute to the spread of adaptive traits, including antimicrobial resistance. Horizontal gene transfer is common in Pasteurellaceae, and aligns with recent mobilome analysis, which highlights the role of mobile genetic elements (including plasmids, transposons and integrons) in shaping the resistome of these pathogens (da Silva et al., 2022). Although none of the plasmids carried antibiotic resistance genes, unlike many clinically relevant plasmids (Watts et al., 2021), this does not preclude their potential to acquire such genes. Recently, several small plasmids have been detected in Haemophilus spp., carrying genes coding for the β-lactamases TEM-1 and TEM-40 (Watts et al., 2021). In this case, our strains were already XDR and had all the resistance determinants integrated in the genome (even the β-lactamase TEM-1 in the case of HUMT-HP05). However, the presence of these plasmids may help transfer these resistance determinants to other bacterial strains. Antimicrobial resistance genes can be incorporated through recombination events, such as the insertion of transposable elements (insertion sequences, transposons) or integrons carrying resistance determinants (Carattoli, 2013). The presence of recombination-associated genes on these plasmids suggest they have the machinery to facilitate such genetic exchanges, consistent with the role of plasmids as genetic reservoirs that can acquire and disseminate resistance genes under selective pressures (Carattoli, 2013).

In conclusion, this study highlights the genetic diversity of H. parainfluenzae and the emergence of multidrug-resistant strains in urogenital infections. The identification of new capsular operons enhancing virulence in XDR isolates, and the acquisition of resistance genes like the bla_CTX-M-15, underscores the evolving threat of this pathogen. While these genomic findings are compelling, functional studies are needed to confirm the contribution of these capsular operons to pathogenicity. Nevertheless, these findings raise concerns about transmission, particularly in MSM populations, and emphasize the need for ongoing genomic surveillance to monitor the spread and pathogenic potential of these strains.