Bacteriophage facilitated transmission of multidrug efflux pump regulatory genes in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a clinically relevant opportunistic pathogen that has been widely detected in various environmental systems, especially in soil and water impacted by intense human activity [1]. It is considered a major healthcare-associated pathogen, often causing infections via contact with contaminated surfaces, medical devices, or water sources [1]. Moreover, P. aeruginosa evades host immune defenses primarily through adhesion, colonization, and biofilm formation [2]. It also produces multiple virulence factors, including exotoxins and proteases, which contribute to tissue damage [3]. In patients with cystic fibrosis, impaired lung epithelial function and reduced phagocytic activity further increase susceptibility to P. aeruginosa infections [3], [4], [5]. Multidrug-resistant P. aeruginosa (MDR-PA) infections, particularly those affecting the lower respiratory tract, are associated with high mortality rates and remain difficult to treat [2]. In China, P. aeruginosa accounts for approximately 16.9–22.0 % of hospital-acquired pneumonia cases, making it the second most common causative pathogen [6]. According to data from the U.S. Centers for Disease Control and Prevention (CDC), 32,600 nosocomial infections caused by MDR-PA were reported in 2017, resulting in an estimated 2700 deaths [7]. These figures highlight the urgent need for in-depth investigation into the mechanisms underlying antibiotic resistance, which is essential for reducing the healthcare burden posed by MDR-PA infections.

The widespread prevalence of P. aeruginosa is largely attributed to its highly adaptable resistance mechanisms, which include the production of inactivation enzymes, reduced membrane permeability, target site modifications, biofilm formation, and the presence of integrons, among others [8], [9], [10]. This versatility has allowed P. aeruginosa to withstand a broad spectrum of antibiotics and antiseptics [11], [12], [13]. Among its most prominent resistance strategies are efflux pumps, such as MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY (OprM), which actively expel a variety of toxic compounds, including antibiotics, thereby decreasing intracellular drug accumulation and enhancing survival [14], [15], [16], [17], [18], [19]. Furthermore, these efflux systems are tightly regulated by specific transcriptional regulatory genes, including mexR, nfxB, mexT and mexZ, which modulate their expression in response to environmental and antibiotic-induced stress. For instance, expression of the MexAB-OprM efflux system in wild-type strains is regulated by the repressor gene mexR [20]. Similarly, MexCD-OprJ and the MexEF-OprN efflux pump expression are regulated by the repressor nfxB [21], and the regulatory genes mexT [22] and mexS [23], respectively. Notably, mexT is critical for the expression of MexEF-OprN efflux pump, as mexT is a transcriptional activator that directly activates the expression of MexEF-OprN efflux pumps, while mexS indirectly negatively regulates the expression of this efflux pump by inhibiting mexT activity [24](Supplementary figure S1). Additionally, bacteriophages (referred to as phages in this work), are viruses that specifically infect bacteria and are closely associated with P. aeruginosa [25], [26], [27], [28], [29]. Phages recognize and bind to bacterial surface receptors, inject their genetic material, lyse the host cell, and release new viral progeny through the lytic cycle [30], [31], [32], [33], [34]. This mechanism forms the basis of their use in targeting bacterial infections. In clinical settings, a wide range of P. aeruginosa-specific phages have been isolated, highlighting their potential as therapeutic agents against MDR-PA infections [27], [35], [36], [37], [38]. Recent studies suggest that phage therapy can serve as an alternative to antibiotics or enhance their efficacy when used in combination [26], [39], [40], [41], [42], [43]. Importantly, phages (whose genome lengths range from 5000 bp to 5000 kb [44]) can mediate the transfer of host-derived genetic material to other bacteria, particularly through temperate phages, thereby promoting horizontal gene transfer (HGT) [45]. Several studies have shown that phages carry a large number of genes (including clinically relevant antibiotic resistance genes), which can be disseminated within microbial communities [45], [46], [47], [69]. This gene transfer mechanism contributes to bacterial evolutionary adaptation and may promote the spread of antibiotic resistance determinants. Although some studies have demonstrated that phages can disseminate virulence factors and antibiotic resistance genes, research specifically addressing these mechanisms in P. aeruginosa remains limited. This represents a critical knowledge gap, as understanding phage-mediated gene transfer in this pathogen could be key to developing novel strategies to manage bacterial resistance.

Here, we hypothesize that multidrug efflux genes and their regulatory genes in P. aeruginosa can be carried by phages, which further mediate the HGT of these regulatory genes. First, we screened for the presence of efflux pumps (MexAB-oprM, MexCD-oprJ, MexEF-oprN) and their associated regulators (mexR/nfxB/mexT) in P. aeruginosa genomes from NCBI database, as well as in viral sequences from the JGI-IMGVR database. We then analyzed the global distribution of phages carrying these genes to identify geographic and environmental hotspots of gene dissemination. Finally, we demonstrate the in vitro transfer of the repressor gene mexR at both cellular and molecular levels using P. aeruginosa strains PAO1/PA1, along with the filamentous phage Pf4. This study provides direct evidence that phages can act as carriers of efflux regulatory factors and proposes a mechanistic model for phage-mediated HGT in clinical P. aeruginosa isolates.