Introduction

In the struggle against bacterial infectious diseases, antibiotic represent the most effective class of medication at our disposal. However, the recurrent development of bacterial antibiotic resistance limits considerably the effectiveness of these agents as well as our therapeutic options. Moreover, the increase of multidrug-resistant (MDR) strains is now a worldwide concern and represents a serious public health issue, recognized by the World Health Organization [39]. Among the most problematic MDR strains are Gram-negative bacteria which produce extended-spectrum β-lactamases (ESBLs) such as P. aeruginosa and Escherichia coli [23].

Depending on bacteria species, resistance to antibiotic can be inherent (constitutive or induced), developed through mutation, and/or received through encoding genetic material from different strains [6]. Thus, bacteria have the ability to adapt and develop mechanism to escape any intrusive product that could undermine the sustainability of the species, and the overuse of antibiotics presumably generates high level of selective pressure which facilitates growth of resistant bacteria [6]. However, bacterial adaption to aggressive compounds suggests a very complex and reactive arsenal of protection. Recent advances in the comprehension of bacterial behaviors demonstrated the presence of cell-to-cell communication mechanism termed quorum sensing (QS) which regulated social bacterial behaviors [19]. Indeed, bacteria are able to detect their population density by producing, releasing, and perceiving small diffusible molecules called autoinducers and allowing them to coordinate a common action implicated in infection success which rely on virulence expression and invasion abilities [7].

Nowadays, evidences have been reported on the implication of QS mechanism in the ability of bacteria to escape antibiotic aggression. For instance, in E. coli, overexpression of SdiA, a LuxR homologue protein that regulates cell division in a cell density-dependent manner [36], confers multidrug resistance and increases levels of AcrAB protein, a component of the antibiotic efflux pump AcrAB-TolC [34]. Moreover, a decreased level of AcrB protein is observed in sdiA null mutants exhibiting consequently a hypersensitivity to fluoroquinolones [34].

P. aeruginosa, an opportunistic Gram-negative pathogen with broad resistance to antibiotics [23] that causes severe infections in immune-compromised hosts [7], shares several mechanisms with E. coli such as antibiotic resistance including efflux systems and β-lactamase production [28] as well as the LuxR/I type QS mechanisms [9]. However, few studies have addressed the correlation between QS mechanisms and bacterial susceptibility to conventional antibiotics in this bacterial model [16, 35]. The aim of the present report is to review relevant literature related to the potential relationship between QS mechanisms and bacterial response to antibiotics in P. aeruginosa.

Overview of QS and Antibiotic Resistance Mechanisms in P. aeruginosa

QS Mechanisms in P. aeruginosa

During the last three decades, P. aeruginosa QS mechanisms and their implication in pathogenicity have been largely studied [2, 21]. Briefly, P. aeruginosa possesses two main QS systems (las and rhl) which drive the production (by the synthetases LasI and RhlI) and the detection (by the transcription factors LasR and RhlR) of the autoinducer signaling molecules N-(3-oxododecanoyl)- l-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-l-homoserine lactone (C4-HSL), respectively [3]. In las system, the diffusible molecule 3-oxo-C12-HSL activates and interacts with the transcription factors LasR when they reach a putative threshold concentration in the cell environment. This interaction drives to the increase of lasI expression and triggers the production of virulence factors including LasB elastase, LasA protease, Apr alkaline protease, and exotoxin A (Fig. 1). Similarly, the interaction of C4-HSL with transcription factors RhlR increases rhlI expression and enhances the production of rhamnolipids, pyocyanin, LasB elastase, hydrogen cyanide, and cytotoxic lectins. The rhl system is also regulated, at the transcriptional and posttranscriptional levels, by the las system in a hierarchical manner [13]. A third QS system, based on the release of a 2-heptyl-hydroxy-4-quinolone [an intercellular signal designated the Pseudomonas quinolone signal (PQS)], interacts with the acyl-homoserine lactones (AHLs) systems in an intricate way [40]. This secondary metabolite of P. aeruginosa is incorporated into the QS hierarchy in times of cell stress and acts as a link between the las and rhl QS systems [8] (Fig. 1). Recently, a fourth intercellular communication signal termed “Integrated QS system” (IQS) has been discovered [20]. This QS system uses new class of quorum-sensing signal molecules (2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde), which have been demonstrated to be able to partially take over the functions of the central las system under phosphate depletion stress conditions. Moreover, it positively regulates the production of PQS and C4-HSL signals, as well as the virulence factors such as pyocyanin, rhamnolipids, and elastase [21].

Fig. 1
figure 1

The four described quorum-sensing systems in P. aeruginosa (AHL-based (las and rhl) systems, quinolone-based (PQS) system, and IQS system). Green arrows indicate a stimulatory effect (Color figure online)

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Mechanisms of Resistance to Antibiotic

Antibacterial drugs present mainly four key mechanisms of action which generate either bactericidal (i.e., killing the bacteria directly) or bacteriostatic effects (i.e., slowing down the reproduction of bacteria) [11]. These mechanisms are mainly based on the inhibition of bacterial cell wall synthesis (e.g., cefotaxime, class of cephalosporin), bacterial deoxyribonucleic acid synthesis (e.g., ofloxacin, class of fluoroquinolone), bacterial protein synthesis (e.g., tobramycin, class of aminoglycosides), or essential metabolites (such as folate) synthesis (e.g., trimethoprim, class of sulfonamides) [12]. The biochemical mechanisms of acquiring resistance to most classes of antibiotics are not completely understood with some exceptions [1]. It is suggested that when first exposed to a new antibiotic, bacteria tend to be largely susceptible and only a few may survive from this antibiotic exposure. Those that survive usually have some genetic characteristic which arise from random mutations, and can be spread among bacteria and transmitted by reproduction to the offspring which become less sensitive to antibiotic by carrying the genetic characteristics of the parent microbe [5]. Drug resistance may also be carried by plasmids or small segments of DNA called transposons, and some plasmids can be horizontally transferred between bacterial cells in a population and between different, but closely related bacterial populations [4].

Antibiotic resistance in P. aeruginosa is mediated via several distinct mechanisms including the production of enzyme that inactivate antibiotics (e.g., production of β-lactamase that degrade β-lactamin antibiotics), the production of efflux pumps (e.g., MexAB-OrpM efflux pumps extruding β-lactamin antibiotics), and the modification and/or alteration of target site or outer membrane (e.g., mutation in gyrA gene encoding the target enzyme of fluoroquinolone antibiotics, a DNA gyrase) [18] (Fig. 2). Beyond the resistance of pathogenic microorganisms to individual antibiotics, P. aeruginosa can develop resistance to multiple antibiotics which is the result association of different mechanism in a single bacteria or the action of single potent mechanism that generate a “cross- or co-resistance” [23]. In this case, Pseudomonas resistance mainly relies on β-lactamase production and overexpression of efflux pumps [37].

Fig. 2
figure 2

Bacterial antibiotic resistance mechanisms [4, 18]. (i) Active drug efflux systems that remove toxic compounds from cells (i.e., resistance to tobramycin and cefotaxime); (ii) mutations resulting in altered cell permeability and decreased penetration of antibiotic (i.e., resistance to ofloxacin); (iii) enzymatic degradation of antimicrobials by the synthesis of specific enzymes for each antimicrobial classes (i.e., resistance to tobramycin and cefotaxime); (iv) alteration/modification of the target site through mutation of key binding elements (i.e., resistance to ofloxacin and trimethoprim)

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QS and Efflux Pump Systems Correlation in P. aeruginosa

Few studies have clearly addressed the linkage between QS systems and antimicrobial susceptibility mechanisms. However, QS was found to be involved in antimicrobial resistance regulation via efflux pump genes [25].

Among different types of antibiotic efflux system in bacteria, the resistance-nodulation-division (RND)-type efflux pumps that use proton motive force as sources of energy are commonly found in Gram-negative bacteria [33]. P. aeruginosa encodes dozen possible RND-type efflux systems [38] among which MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM have been characterized [30, 31]. Efflux transporters are organized as tripartite systems where the pump located in the inner membrane (e.g., MexB, MexD, MexF, and MexY) works in conjunction with a periplasmic protein (e.g., MexA, MexC, MexE, and MexX) and an outer membrane protein (e.g., OprM, OprJ, and OprN). Functioning of each efflux pumps lead to extrusion of broad substrates including antibiotic and chemotherapeutic agent as well as AHLs with acyl chain lengths of C8–14 including 3-oxo-C12-HSL as summarized in Table 1. Indeed, in contrast to C4-HSL and 3-oxo-C6-HSL which are known to diffuse freely in and out the bacteria [15, 29], 3-oxo-Cn-HSLs with long acyl chain lengths of C8–14 requires active transport through the MexAB-OprM and MexEF-OprN efflux systems [15, 27]. Secretion of AHLs through MexXY-OprM and MexCD-OprJ has not been yet documented.

Table 1 Substrate specificities of MexAB-OprM, MexEF-OprN, MexCD-OprJ, and MexXY-OprM efflux pumps in P. aeruginosa (unexhaustive list)
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In regard to the relation between efflux and QS systems, Evans et al. [10] demonstrated that P. aeruginosa mutant strains overexpressing the mexAB-oprM exhibit reduced expression in lasI and produce less 3-oxo-C12-HSL and virulence factors (including pyocyanin, elastase, and casein protease) compared to wild-type strains. Likewise, Köhler et al. [31] reported that mutants overexpressing the mexEF-oprN efflux system exhibit reduced expression in rhlI and produce lower levels of C4-HSL and extracellular virulence factors (pyocyanin, elastase, and rhamnolipids) as compared to wild type. Interestingly, Maseda et al. [25] demonstrated that exogenous addition of the C4-HSL enhanced the expression of mexAB-oprM operon in laboratory-derived PAO4290 strains, whereas 3-oxo-C12-HSL had only a slight effect. This study also revealed that MexAB mutants accumulate 3-oxo-C12-HSL intracellularly which suppose a QS-efflux pumps correlation. The correlation between AHLs level production and efflux pumps expression (mexAB-oprM and mexEF-oprN) in P. aeruginosa are summarized in Fig. 3.

Fig. 3
figure 3

Interaction between QS and efflux pump systems in P. aeruginosa. Fact 1 overexpression of mexAB-orpM leads to increase of antibiotic extrusion but also to an important extrusion of 3-oxo-C12-HSLs and consequently decreases the formation of 3-oxo-C-12-HSL-LasR complex and virulence factors expression. Similarly, overexpression of mexEF-orpN leads to a decrease in rhlI expression and C4-HSL concentration [17]; consequently, high levels of antimicrobial resistance with reduction in virulence factor expression are recorded. Fact 2 Exogenous C4-HSL supply enhances expression of mexAB-orpM (Maseda et al.) [25] that presumably extrudes specific antimicrobials but could also extrude noncognate 3-oxo-Cn-HSLs [27] and consequently optimizes formation of 3-oxo-C-12-HSL-LasR complex leading to high QS-regulated and QS-related genes expression [13]. Fact 3 Lack of MexAB-OrpM pumps leads to an intracellular accumulation of 3-oxo-C-12-HSL that could limit cell-to-cell communication in P. aeruginosa

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Recently, Pourmand et al. [32] reported a positive correlation between mexXY-oprM expression and las system among P. aeruginosa clinical strains isolated from wound infection where strains with increased lasI and lasR gene expression exhibit higher mexY gene expression and isolates with low lasI and lasR expression shown decrease mexY gene expression.

QS Regulation and Bacterial Sensitivity to Antibiotics in P. aeruginosa

In view of studies reported above, we are tempted to presume that the QS mechanism could influence bacterial susceptibility to antibiotics. Intriguingly, Karatuna and Yagci [16] reported that P. aeruginosa clinical isolates obtained from lower airways clinical samples that were deficient in QS genes (detected through PCR amplification by using oligonucleotide primers designed for lasI, lasR, rhlI, and rhlR genes) were generally less susceptible to antibiotics (including piperacillin, ceftazidime, tobramycin, and ciprofloxacin assessed by disk diffusion method) compared to P. aeruginosa PAO1. However, these clinical strains may harbor inherent or acquired antibiotic resistance mechanisms that have not been evidenced during experiments. Intriguingly, Rampioni et al. [35] demonstrated the importance of RsaL, negative regulator of lasI expression, in P. aeruginosa resistance to antibiotics through efflux pump systems modulation. Indeed, antibiotic susceptibility to trimethoprim, chloramphenicol, norfloxacin, and nalidixic acid is enhanced in rsaL mutant with respect to the wild type, and the microarray analysis showed that rsaL mutation leads to downregulation of mexEF-oprN multidrug-efflux system operons. However, authors also demonstrated that RsaL works as global regulator per se, independently of its repressive effect on lasI expression. Indeed, RsaL control at least 341 genes, including genes involved in biofilm formation and antibiotic resistance which could contribute to the increased sensitivity of rsaL mutant strain to antibiotics. Thus, implication of AHLs-based QS system and bacterial response to antibiotics are not clearly established. In our opinion, to better understand the link between QS mechanism regulation and bacterial response to different antibiotics, several experiments should be conducted. For instance, sensitivity to antibiotics should be evaluated in two different conditions where QS mechanism in PAO1 is altered. Such condition could be achieved by adding exogenous AHLs to PAO1 culture medium and by using PAO1 altered by mutations, specifically in LasI/R and RhlI/R systems. Moreover, to bring light on the hierarchical importance of QS systems and to precise the implications of QS in efflux pumps systems regulation, expression of efflux pump genes (e.g., mexA, mexB), and their corresponding regulator genes in Las and Rhl mutant strains as well as influence of both AHLs on efflux pump systems (e.g., mexAB-orpM) should also be assessed. These investigations are necessary to determine the precise role of QS actors (AHLs, regulator proteins, and QS genes) in antibiotic susceptibility and theirs interaction with conventional antibiotics.

Concluding Remarks

It is well-admitted that bacteria have the ability to adapt and develop mechanism in response to any intrusive compounds that could impair sustainability of its species [6]. In the case of antibiotic, its overuse presumably precipitate increase rate of resistant bacteria [9]. On one hand, an important potential strategy to resolve this issue is the development of new active agents capable to suppress bacterial resistance mechanisms [24]. On the other hand, research for anti-QS has been largely explored since last decades, in order to propose new alternative to struggle against bacterial infection with a limited selective pressure [14]. The present paper highlights that QS probably contribute to optimize some of the arsenals developed by bacteria to escape antibiotics aggression by upregulation of efflux pump genes. However, we have to admit that this interconnection remains nebulous and the hierarchical importance of QS systems in this resistance process needs to be investigated. While it is recognized that efflux pump systems represent an important “pivot” for antimicrobial resistance, AHLs management and diffusion, further investigation should be carried out to bring strong arguments for the importance of QS in the bacterial adaptation to antimicrobial aggression and thus, put QS as efficient anti-infective target. Furthermore, impact of RsaL on the modulation of P. aeruginosa antibiotic susceptibility eggs on investigating the impact of other negatives (e.g., global posttranscriptional regulator, RsmA and quorum-sensing control repressor, and QscR) and positive (e.g., GacS/GacA two component system and CRP-homologous regulator, and VfR) QS global regulator. Finally, studying interconnection between QS system and efflux system connection in antibiotic response should be also extended to the non-AHL-based QS system (PQS system and the recently known IQS) as they could represent the missing link in the hierarchical quorum-sensing system circuitry in P. aeruginosa.