Characteristics and clinical relevance of Pseudomonas aeruginosa

The strictly aerobic rod-shaped Gram-negative Pseudomonas aeruginosa bacteria can be frequently isolated from individuals suffering from chronic lung illness such as patients with cystic fibrosis (CF) and chronically recurrent infections are further perpetuating pulmonary diseases leading to bronchiectasis, fibrosis, and lethal lung failure [1]. While the inflammatory changes of the affected tissues are aggravated by bacterial exotoxin A as well as proteases and elastases, biofilm formation by P. aeruginosa in particular imposes a challenge for pathogen eradication by host immunity and antibiotic treatment [1].

P. aeruginosa infections mostly establish in hospitalized patients, and particularly in ventilated patients treated on intensive care units (ICUs) the opportunistic pathogens are often responsible for nosocomial pneumonia. In general, infections with P. aeruginosa frequently engender bacteremia, and the initial evidence of P. aeruginosa infection in hospitalized patients occasionally manifests first by sepsis [2].

According to the Centers for Disease Control and Prevention (CDC), there were roughly 32,600 infections with multidrug-resistant (MDR) P. aeruginosa in US hospitals in 2017, resulting in approximately 2,700 deaths and $767 millions healthcare costs [3]. Some individual bacterial strains exhibit resistance to almost all available antibiotics. The high prevalence of carbapenem resistance in the P. aeruginosa population is of particular clinical concern, and up to 3% of resistant bacterial strains possess a transposable element, which allows for rapid exchange, distribution, and dissemination of carbapenemase production among bacterial communities [3].

Therefore, MDR P. aeruginosa are listed as serious threat to human health in the CDC's 2019 Antibiotic Resistance Threats Report [3]. In addition, the World Health Organization (WHO) records carbapenem-resistant P. aeruginosa in its list of the most important antibiotic-resistant bacteria requiring research and development of new antibiotics in the highest category of priority, indicating critical urgency [4].

Another therapeutic approach to multidrug resistance

According to the UN Ad hoc Interagency Coordinating Group on Antimicrobial Resistance it is estimated that in the worst case scenario up to ten million deaths per year worldwide will be attributable to MDR infections by 2050 [5]. Bacteriophages, viruses that specifically tackle bacteria, represent a possible part of the solution to this threat to humankind.

Initially, as first comprehensively described by the French microbiologist Félix d’Hérelle in 1917 [6, 7], phages were widely used to combat bacterial infections as early as the 1930s, when in parallel the first antibiotics (arsphenamin (Salvarsan™) and sulfonamides) were on the market [8]. By now, not only the whole phage particles, but also the endolysins produced and released by them at the end of the lytic replication cycle are assigned clinical importance since these enzymes exert antimicrobial effects by damaging the bacterial cell walls [9]. Moreover, phages as well as their lysins also appear to be principally effective against (and even in spite of) bacterial pathogens located in biofilms [10].

Although phage therapy is non-toxic and has several favorable characteristics, such as efficacy against resistant bacteria, lack of harm to the resident commensal microbiota and self-regulating dosage, it is still most uncommon in modern medicine of high-income countries, especially in the Western world [11]. Depending on the focus of infection, different sides and means of phage administration may be considered, as well as the use of certain combinations of phages, called “phage cocktails” [12].

Aim

The aim of this review is to bring together the findings of various studies on the efficacy and effects of phages against P. aeruginosa in different contexts, in order to gain an overview of the (gaps in) evidence for their potential suitability to counter distinct lung infections caused by MDR strains of the pathogens in particular.

Inclusion and exclusion criteria

Prerequisite conditions of inclusion were a publication date within the last ten years and the publication's language being English. Original publications studying phages as potential therapeutic agents against P. aeruginosa induced pulmonary infections were included in this literature survey. Given the heterogeneity of the existing literature, we included studies performed in silico and on endolysins of phages as well as case reports. Since there was only a limited number of relevant publications, no further specifications were imposed. Subject of exclusion were studies focusing on other species of the genus Pseudomonas than P. aeruginosa or research methodology.

Search strategy and data extraction

The search for pertinent literature was undertaken on the 18th of May 2023 by using the search engine “PubMed” by the United States National Library of Medicine accessing the MEDLINE database. The exact procedure of using the “PubMed Advanced Search Builder” is shown in Table 1. Synonyms, tagged as “Title/Abstract”, within the individual queries were addressed by inclusive disjunctions through the Boolean operator “OR”. The eventual search was composed of the five preceding parts with all linked together by employing the Boolean operator of conjunction “AND” resulting in 29 publications, of which four reviews, according to the respective tag “Publication Type”, were excluded through the Boolean operator of negation “NOT”. Figure 1 summarizes the whole process of the study selection ultimately resulting in 23 publications as the basis for this review.

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Schematic depiction of the selection process of publications

Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00060

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Inhibition and killing of P. aeruginosa and effects beyond

In their study, Chang et al. tested the phage PEV20 administered by inhalation as a dry-powder spray with lactose and leucine in neutropenic mice that had been infected with P. aeruginosa FADDI-PA001 via the trachea [13]. Two hours post-infection, mice were intratracheally challenged with 2 mg of the suspension containing 4 × 10⁷ phage particles as determined by enumeration of plaque-forming units (PFU). In comparison to a control group, a reduction in the bacterial burden of the lungs by 5.3 orders of magnitude was ascertained at 24 h after onset of therapy. Moreover, there was an increase of the phage's pulmonal presence by one order of magnitude. In terms of safety, neither alveolar epithelial nor macrophage cells were affected by the PEV20 powder in vitro and an additional assessment of safety conducted through histological investigation showed only slight harm. Phages administered as a therapy for P. aeruginosa lung infection were bound up with a mitigation of lung damage, however [13].

Yang et al. investigated the capacity of phages to inhibit growth of P. aeruginosa in vitro and additionally, their therapeutic effects on hemorrhagic pneumonia in mice [14]. Firstly, the authors isolated P. aeruginosa D9, which turned out to be resistant to a wide range of antibiotics, from minks having presumably died due to hemorrhagic pneumonia. For the in vitro experiment, prepared P. aeruginosa D9 and the phage YH6 were added to a double strength lysogeny broth. Then, the compound was thinned to normal-strength lysogeny broth with a bacterial number of 10⁸ colony-forming units (CFU)/mL. The addition of phages was carried out separately at a multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 1.0. For MOI = 0.01 and MOI = 0.001 at one hour after incubation, no meaningful effects of exogenous phages on bacterial growth were detected. On the other hand, a rather fast and tremendous drop-off of optical density at a wavelength of 600 nm (OD₆₀₀) values occurred at the MOIs of 0.1 and 1.0. From the tenth hour after incubation on, an essential reduction in OD₆₀₀ was mutually evident for all groups of bacteria infected by phages indicative for enhanced bacterial killing. As part of the in vivo experiment, mice received an intranasal injection of 2 × 10⁸ CFU of P. aeruginosa D9 (30 μL volume), equivalent to the double minimum lethal dose. Following this, the different groups were given 2 × 10⁵, 2 × 10⁶, 2 × 10⁷, or 2 × 10⁸ PFU of the phage YH6 for treatment by the nasal route at two hours post-infection. A control group received phosphate-buffered saline (PBS) as placebo only. Administering a dosage of 2 × 10⁵ PFU did not enhance the survival rate of mice, however. Every mouse that received a dosage of 2 × 10⁷ PFU of the phage fully recuperated without displaying observable symptoms, and the macroscopic aspects of the lung tissues were comparable to those in healthy mice. What is more, the P. aeruginosa loads in the blood and lungs decreased by about 2.6 and 3.0 orders of magnitude, respectively, upon phage treatment until 24 h post-infection, whereas in the same time period, the median bacterial counts increased by more than 3 orders of magnitude in both, the blood and lungs of mice from the PBS control cohort [14].

Chow et al. surveyed the pharmacokinetics of a phage therapy against P. aeruginosa administered via the airways in neutropenic mice [15]. Initially, a group of mice was given 10⁷ and 10⁹ PFU of the phage PEV31. Within 24 h, the phage loads decreased to 12.3% and 15.2% of the originally applied numbers, respectively. Independent of the doses, a half-life period of about eight hours was observed. Less than one ten-thousandth of applied dosages was found in total in kidneys, livers, spleens, and blood. Of note, an increase of the hepatic phage titer was shown in one day's time after the application of 10⁹ PFU of the phage PEV31 that was associated with an increased expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) at four- and eight-hours’ time as measured in bronchoalveolar lavage (BAL) fluid. In another experiment, the authors inoculated mice with MDR P. aeruginosa and provided a treatment with 10⁹ PFU of the phage PEV31 two hours later. Consequently, the median pulmonal phage numbers increased by approximately two orders of magnitude at 24 h after adding the phages due to lytic replication in target bacteria, which further suppressed Pseudomonas growth. Whereas the mock-treated control group with full-blown bacterial infection displayed enhanced expression of pulmonary pro-inflammatory cytokines, the upregulation of interleukin-1 beta (IL-1β), but neither of TNF-α nor of IL-6 was partially suppressed in the BAL fluid upon phage treatment. Another finding of the study was that in some of the treated mice, the antibiotic susceptibility patterns of the isolated P. aeruginosa bacteria had changed within 26 h post-infection [15].

Chen et al. performed in vitro and in vivo experiments with selected phages against P. aeruginosa [16]. In plaque testing against 24 clinical and 7 laboratory isolates of P. aeruginosa, the phages MYY9, HX1, and MYY16 exhibited host infectivity rates of 87.1%, 87.1%, and 71.0%, respectively. In an in vitro experiment testing their bactericidal capabilities, the phages HX1 and MYY9 were able to interfere with the P. aeruginosa PA01 strain at an MOI of 0.000001, whereas MYY16 caused no additional reduction in PA01 at an MOI of 0.01 compared to the control group. In order to test the phages in acute murine pneumonia, mice were infected with P. aeruginosa PA01 strain and were then subjected to the phages MYY9 and HX1. A reduction in bacterial burdens of about 3.2 log units was observed in case of intratracheal application of each phage. Injection via the tail vein led to a reduction in P. aeruginosa burdens of 1.25 log units and 0.57 log units in case of the phages MYY9 and HX1, respectively. Furthermore, histopathological examinations revealed less distinct lung damage in phage treated P. aeruginosa infected mice, whereas no pulmonary inflammatory signs were detected in case of exclusive phage therapy (i.e., in non-infected mice). The mucoid P. aeruginosa isolate FRD1 from CF sputum was used as a murine model for chronic pneumonia. Application of the phage MYY9 provided evidence for an anti-pseudomonal effect in vivo given a decrease of P. aeruginosa counts of 4.7 log units four days after infection that was accompanied by a decline of the phages after lysis. Additionally, the use of MYY9 was associated with lower levels of TNF-α and IL-6 as measured in pulmonal explants [16].

Zhang et al. performed experiments with the phage vB_PaeP_PA01EW and investigated its potential as an anti-Pseudomonas directed therapeutic agent [17]. When using an in vitro infection assay, a P. aeruginosa PA01 strain growth inhibition was observed in presence of the phage vB_PaeP_PA01EW at MOIs of 0.01 and 0.1. Furthermore, the phages demonstrated distinct temperature resistance at 4 °C, 25 °C, and 37 °C. For the in vivo study, two groups of mice (among others) were inoculated with 10⁶ CFU of P. aeruginosa PA01 via the trachea and received either plain saline (mock group) or 10⁹ PFU of the phage vB_PaeP_PA01EW (all at an identical volume of 50 µL) at one hour post-infection. In comparison to the mock controls, an attenuation of edematous adventitia and tissue dissemination of inflammatory cells was shown in the phage treatment cohort. Additionally, a substantial decrease of pulmonal P. aeruginosa loads was observed, underlining the phages' lytic ability against the PA01 strain in vivo. Nonetheless, epithelial proliferation of alveolar cells and occasional bronchial invasion of immune cells were reported despite phage therapy. Hence, the authors expected an even better clinical outcome if the phages were used concomitantly with antibiotics [17].

Lytic ability of phages under different conditions

Chang et al. examined the potential dose-dependent effects of an inhalative phage therapy in mice [18]. Therefore, neutropenic mice were exposed to 2 × 10⁴ CFU of MDR P. aeruginosa FADDI-PA001 via the trachea. Two hours post-infection, three groups of infected mice received a suspension of the phage PEV31 (25 μL) in PBS as treatment via the tracheal route, each at varying doses; namely Group A with 7.5 × 10⁴ PFU, Group B with 5.0 × 10⁶ PFU, and Group C with 5.0 × 10⁸ PFU. A control group received vehicle only. In the control group, the authors found an almost 3.5 orders of magnitude increase in pulmonal P. aeruginosa loads (i.e., from 7.4 × 10⁴ CFU at two hours after infection to 2.3 × 10⁸ CFU at 24 h after infection). The application of the phage therapy, however, resulted in an 1.3 to 1.9 orders of magnitude decrease in lung bacterial loads as shown by the following mean CFUs at 24 h post-infection: 1.1 × 10⁷ CFU in Group A; 6.5 × 10⁶ CFU in Group B; and 3.1 × 10⁶ CFU in Group C. In addition, decreases in the P. aeruginosa loads were observed in the kidneys, the spleens, and even systemically in the blood following phage treatment. Interestingly, both the highest absolute PEV31 counts as well as the most pronounced (relative) increase in phage numbers were detected in Group A within 24 h. While mice in Group B also exhibited an enhanced PEV31 titer indicative of a vivid phage replication, this could not be observed in animals of Group 3. Instead, in 91% of mice a resistance of P. aeruginosa against the applied phages could be observed, whereas this held true for 30% and 74% of cases in Group A and Group B, respectively. The authors hypothesized that the fast killing of phage-sensitive bacteria through high doses might promote the proliferation of the phage-resistant bacteria, whereas progressive killing constitutes a prerequisite for the re-growth of phage-sensitive bacteria by the expenses of phage-resistant bacteria. Associated with the emerging PEV31-resistant bacteria was an enhanced susceptibility to antibiotics. Pulmonal expression of pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α were highly up-regulated in mock control mice, whereas respective mediators were down-regulated in the treatment groups in a dose-dependent fashion [18].

Li et al. tested a mixture of three phages as therapy in dispersed powder form against respiratory P. aeruginosa infections [19]. Fundamental findings prompted to ensure relative humidities below 60% to thwart the powder's crystallization as well as temperatures below 20 °C for the purpose of proper storage. The absence of unfavorable phage-phage interactions underscored the suitability of powder form in principle. While the phages PEV2, PEV1, and PEV20 experienced a decrease of their titers by 0.1, 1.3, and 0.7 log orders of magnitude, respectively, due to the process of drying, no functional impairments could be observed in the phages. Hence, the phage cocktail was able to successfully lyse the P. aeruginosa JIP125, JIP117, and JIP62 strains in vitro. Hence, the authors suggested the potential use of phage cocktails as powder against coincident infections with P. aeruginosa of different strains [19].

Alvi et al. examined the phage RLP, which exhibited a host infectivity rate of 50% in case of the 38 P. aeruginosa strains tested [20]. For an in vivo experiment, mice were infected with P. aeruginosa PA-1 at the minimum lethal dose. Whereas only 7.4% of the mice without phage therapy survived, this was the case for 92% of mice following phage RLP application. Furthermore, pseudomonal growth was inhibited in vitro at an MOI of 10 until 20 h post phage RLP challenge. While its pH optimum appeared at 7.0, its logarithmic titer was reduced by factor five at pH 5.0 and pH 9.0. Furthermore, 25 °C and 4 °C could be considered as appropriate incubation and storage temperature conditions for RLP, respectively [20].

Endolysins derived from phages

Guo et al. investigated the antibacterial impact of the endolysin LysPA26 derived from the phage JD010 on P. aeruginosa in vitro [21]. The findings indicated, in general, an inherent antibacterial capacity of LysPA26, which was further studied in dependence on temperature, pH, and sodium chloride (NaCl) concentrations. The authors found that a temperature range from 37 °C to 50 °C, a pH of 8.0, and NaCl concentrations below 150 mM were ideal conditions for the endolysin's activity. The LysPA26 phage lysin exerted its bactericidal activity against P. aeruginosa D204 in a dose-dependent manner, although it was noted that a concentration above 0.5 mg mL⁻¹ in the assays was not associated with an advanced lytic effect. However, if added to 10⁸ bacterial cells, the lysin at a dose of 0.5 mg mL⁻¹ was able to reduce the bacterial numbers by 4 log units within half an hour. Aside from that, the findings point to an increase of LysPA26's biological activity in presence of 1 mM ethylenediaminetetraacetic acid (EDTA), as 1 log unit of bacterial cells was lysed additionally. For a test of LysPA26's capacity to destruct biofilms, the lysin was added to P. aeruginosa 8328 strain 48 h following biofilm formation. The authors found that 100 μg of LysPA26 resulted in a decrease of bacterial cells in biofilms by up to two orders of magnitude [21].

In their study, Euler et al. addressed the bactericidal capacities of the phage lysin PlyKp104 [22]. In an in vitro killing assay, co-incubation of P. aeruginosa strain PA01 with 25 μg mL⁻¹ of the phage lysin resulted in a decrease in P. aeruginosa numbers by more than 4 orders of magnitude within 1 h. Additionally, the PlyKp104 lysin was able to reduce further tested P. aeruginosa strains (namely, strain 439-441 and strains 467–471) by 3–4 log units. The phages's lytic capacity against P. aeruginosa PA01 was also tested under varying conditions. Between pH 5.0 to pH 10.0, for instance, the PlyKp104 lysin exhibited strong lytic activity. Further in vitro experiments were performed with pH 7.4, however. When testing for salt tolerance, the lysin was fully effective at 0–500 mM of NaCl. Furthermore, lung surfactant (0–25% [vol/vol]) was proven not to affect the PlyKp104 lysin. Whereas the phage's lytic capacity was severely impaired at a 12% human serum concentration, the PlyKp104 lysin was able to reduce P. aeruginosa numbers by more than 5 orders of magnitude at a 6% human serum concentration. Given the latter observation, the authors excluded therapeutic effects upon systemic application in case of bacteremia [22].

Raz et al. tested the effects of the phage lysins PlyPa03 and PlyPa91 under the influence of various factors [23]. Fundamentally, these two lysins achieved an eradication of P. aeruginosa PA01 by more than 5 orders of magnitude. When assessing pH-dependent effects, bacterial loads could be effectively reduced with the pH ranging from 5.0 to 10.0. Furthermore, the phage PlyPa03 lysin was hardly affected by NaCl concentrations of 500 mM. As opposed to the PlyPa03 phage, PlyPa91 was inhibited and did not show an increased lytic impact upon co-incubation with 0.5 mM EDTA. While already 0.375 mg mL⁻¹ of the PlyPa03 lysin were sufficient for a pronounced destruction of P. aeruginosa biofilms, 1.5 mg mL⁻¹ of PlyPa91 lysin were necessary. Nevertheless, PlyPa91 lysin maintained its lytic capacity when combined with less than 8% human serum, whereas 1% of human serum entirely suppressed PlyPa03. Hence, PlyPa91 lysin is considered a better fit for application in an area with slight quantity of serum. Neither PlyPa03 nor PlyPa91 lysins were impaired in tests with a combination of bovine lung surfactants and artificial surfactants concentrated as high as 25%. Further experiments suggested the incapability of lysing human red blood cells or HL-60 neutrophils. In a murine lung infection model, intranasally and intratracheally applied PlyPa91 lysin led to the survival of 70% of mice at day 10, while all mice without treatment died within 48 h post-infection [23].

Efficacy of phages against pseudomonal biofilms

Fiscarelli et al. conducted an in vitro study addressing the ability of phages to combat P. aeruginosa even in biofilms [24]. The authors gained 22 phages originating from different sources and tested their lytic activities against 32 isolates of P. aeruginosa derived from CF patients. Overall, the lytic effects of the phages under investigation were dependent on the experimental conditions and the clinical origin of the P. aeruginosa isolate. At maximum, the tested phages caused a lysis of 93.7% of the P. aeruginosa isolates. In particular, five phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) were found to feature an extended spectrum of targeted hosts. Beyond that, only the phage Φ4_ZP1 if combined with meropenem exhibited an increased effect of biofilm reduction (in comparison to an exclusive application of meropenem) after four hours when tested against the P. aeruginosa isolates Pa_Ph10 and Pa_Ph12 at MOI 100 and MOI 1, respectively. In combination with tobramycin, the phage Φ14_OBG showed a more pronounced anti-biofilm effect towards Pa_Ph3 and Pa_Ph7 at MOI 100 each, when compared to tobramycin alone. However, the testing of the five phages on nine non-mucoid P. aeruginosa isolates derived from biofilms of CF patients led to the appearance of 30 novel mucoid morphotypes. Besides, bacterial growth returned 24 h after the phages had been applied to clinical P. aeruginosa isolates and an excessive build-up of biofilms was reported [24].

Waters et al. tested the phage PELP20 against P. aeruginosa LESB65 and NP22_2 isolates [25]. In a murine experiment, a chronic lung infection could be completely overcome when the phage therapy was started at 24 h and 36 h or 48 h and 60 h after infection. Even when therapy had not been initiated until day six post-infection, 70% of the mice experienced the elimination of pulmonal bacterial loads, whereas the remaining mice underwent at least a pronounced decrease in bacterial burdens. In another experiment, the authors employed an artificial sputum medium biofilm model mimicking distinct features in CF patients and showed a decrease of P. aeruginosa within biofilm by 3 orders of magnitude within 24 h after use of the phage PELP20. This being the case, the authors inferred a biofilm-penetration property allowing lytic efficacy against the bacteria under mock CF lung conditions [25], which is illustrated by Figure. 2.

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Phages penetrate biofilms and induce their degradation. On the left side, the normalization of a cross-sectional airway after administration of phages targeting the bacteria is illustrated. To achieve such a clinical improvement, phages need to undergo multiple lytic cycles involving the following steps (as depicted by the middle box): attachment to the cell; insertion of genetic material; production and distribution of new phages through lytic release. Additionally, the destruction of respective biofilms is achieved. Since the exact process of the latter is not yet fully understood, the general idea of its effect is only sketchily outlined on the right

Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00060

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Combination of phages and antibiotics

Duplessis et al. analyzed the anti-pseudomonal effects of a phage cocktail comprising PaAH2ΦP, PaBAP5Φ2, and PaΦ. All of the phages were able to inhibit the growth of the P. aeruginosa UNC-D strain alone. While PaBAP5Φ2 maintained bacterial growth inhibition for 22 h, pseudomonal proliferation was impeded for up to 45 h when combined with the other two phages. The same duration of growth inhibition was achieved when the former phage was combined with meropenem. In their in vivo lung infection model, the survival of lethally infected mice was achieved when the phage cocktail was applied via the pulmonary route and intraperitoneally or intubation-mediated via the trachea within three and six hours post-infection, respectively. Exclusive intraperitoneal treatment, however, primarily led to a reduction in pseudomonal loads in the lungs. In combination with subcutaneously injected meropenem, the intraperitoneally administered phage cocktail resulted in a survival rate of more than 50% of lethally infected mice [26].

In their study, Martin et al. addressed the changes of P. aeruginosa's sensitivity to bacteriophages and antibiotics over time. The authors derived 112 P. aeruginosa isolates from 24 CF patients (in average four bacterial isolates per patient) suffering from chronic infection over a period of eight to twelve months. 102 (91%) of the isolates came with dual profiles of sensitivity and data on variable number tandem repeats. When defining MDR as a resistance of the bacterial isolate to at minimum one antibiotic each in at least two antibiotic classes, the authors classified 51 (50%) of the clinical P. aeruginosa isolates as MDR. Yet, 40 (78%) of the isolates showed sensitivity to neat phages and 26 (51%) to diluted phages (10⁻³ dilution). In ten patients, P. aeruginosa strains with a single variable number tandem repeat were isolated and shown to be stably sensitive to neat phages, whereas one patient's isolates remained resistant over time. More pronounced variability was noted when it came to sensitivity to diluted phages. The combination of phages and antibiotics considered, the isolates showed notable variability in susceptibility over time. For instance, a pronounced variability in sensitivity of the bacterial isolates towards tobramycin and meropenem was found in 14% and 29% of 21 patients, respectively [27].

Interactions between phages and host immunity

Dan et al. examined immunological parameters of a patient receiving phage therapy (intravenously and by inhalation) due to pneumonia caused by P. aeruginosa which required a sedated and ventilated state [28]. The lung transplant recipient's comprehensive phage therapy (including, among others, AB-PA01, a composition of four lytic phages, and AB-PA01-m1) led, ultimately, to ambulatory condition without complete bacterial clearance. Within the course of treatment, circulating T follicular helper cells showed peak concentrations and maintained elevated levels, whereas healthy control subjects who were not administered phages exhibited only very few cell counts. In addition, CD4⁺ T cells specific for AB-PA01 and AB-PA01-m1 could be detected that were accompanied by remarkably increased pro-inflammatory cytokine concentrations. Furthermore, immunoglobulin G (IgG) against the two phages were shown to increase 1000-fold in the patients if compared to healthy subjects without phage treatment. Also, newly emerged neutralizing antibodies against various phages were detected. Nevertheless, the authors considered this phage therapy an overall success, given that the patient fully recovered [28].

Weissfuss et al. studied the immune responses following application of a phage cocktail (comprising DSM 19872/JG005 and DSM 22045/JG024) to naive mice [29]. The authors were able to show that phages injected intraperitoneally reached the systemic circulation of mice within six hours without inducing an increase of polymorphonuclear neutrophils in the blood. Furthermore, neither did the splenic numbers of dendritic cells expressing major histocompatibility complex (MHC) class II nor did their mean fluorescence intensity increase following intraperitoneal injection of the phages. However, both, higher CD4⁺ T cell and lower CD8⁺ T cells counts were detected at six hours after phage injection, whereas the overall T cell proliferation remained unchanged. While neither Th1 cell nor regulatory T cell numbers were elevated at 6 h and 10 days, a notable increased systemic secretion of distinct chemokines and cytokines including CXCL10, CCL2, CCL5, and IL-1β were measured at 6 h after application of the phage cocktail. Furthermore, a slight splenic formation of germinal centers indicative for humoral immune responses, and an insignificant increase of IgG were observed on days 10 and 24 post phage challenge. Notably, no adverse effects due to phage application could be assessed. In summary, the authors concluded that phage cocktails constitute promising antibacterial treatment options for P. aeruginosa infections in the future [29].

Roach et al. addressed the role of host immunity during phage treatment of murine lung infections caused by MDR P. aeruginosa [30]. The results obtained from in vivo phage treatment and in silico modeling experiments provided evidence that for both, the successful cure and the prophylaxis of acute P. aeruginosa induced pneumonia in mice, the synergistic interplay between the applied phages on one side and the neutrophilic granulocytes on the host side was essential. Notably, the neutrophils were required to control phage-sensitive as well as phage-resistant bacteria emerging during phage treatment or prophylaxis of acute pneumonia. Based upon their results the authors proposed a novel “immunophage synergy” concept. Furthermore, the therapeutic phages were shown not to be cleared by the immune effector cells of the lung. The authors suggested to refrain from phages as treatment option of P. aeruginosa pneumonia due to an infection in neutropenic patients [30].

In their in vitro and in vivo studies, Abd El-Aziz et al. tested the effects of the phage MMI-Ps₁ in P. aeruginosa induced lung infection [31]. Firstly, the authors inoculated mice with P. aeruginosa PA9 via the nasal airway. While a control group (group 3) was given PBS, the remaining groups were treated with MMI-Ps₁ either immediately (group 1) or twelve hours (group 2) after infection. At 48 h post-infection, approximately 60% of the mice in the control group succumbed to the infection and had to be euthanized, whereas all mice from the MMI-Ps₁ cohorts exhibited only minimal or even no signs of disease at this time point. Furthermore, mice receiving phage therapy directly or at 12 h post-infection demonstrated either no or only subtle bacteremia within 24 h and 72 h following infection. Conversely, the control group's mice presented with substantially elevated bacterial loads in the blood. For their in vitro experiment, the authors tested 40 P. aeruginosa strains regarding their mucoid status and their susceptibility to the MMI-Ps₁ phages. The obtained results revealed that 21 of the 40 strains (52.5%), of which eleven were mucoid and ten non-mucoid, were sensitive to the applied phages. When the P. aeruginosa PA9 strain was co-incubated with 40% human serum for 30 min, there was no observable activity of serum killing. However, when the same strain was incubated exclusively with phages, a notable reduction in the number of viable bacterial counts became apparent. Apart from that, when phages were brought into the combination of serum and bacteria, it fostered the serum killing activity. On this basis, the authors postulated a synergistic effect of phages and complement system on the lysis of MDR P. aeruginosa [31].

Menon et al. explored the properties of hyperpigmented P. aeruginosa mutants, which developed resistance to applied phages [32]. To address this, the authors administered the phages AM.P2, Mat, and Kat to P. aeruginosa PA01 resulting in resistant mutants designated P. aeruginosa AM.P2-X-1, Mat-X-1, and Kat-X-2. The resistance of the phages was due to 300 kb deletions affecting 227 coding regions, as determined by whole-genome sequencing. This loss of genetic information impaired a plethora of virulence factors, albeit corresponding with an excessive production of pyomelanin, which itself is considered an important virulence factor of P. aeruginosa. Indeed, it was found that the mutated P. aeruginosa cells exhibited reduced minimum inhibitory concentrations (MICs) for the human cathelicidin LL-37 and colistin by factors 16 and 4, respectively. The killing of the pyomelanogenic mutants in whole blood or serum turned out to be notably faster compared to the phage-sensitive PA01. Furthermore, the P. aeruginosa AM.P2-X-1 mutant strain survived within the lung of infected mice with 10-times lower counts if compared to P. aeruginosa [32].

Clinical application of phages

In their case report, Law et al. presented a female 26-year-old CF patient suffering from MDR P. aeruginosa induced pneumonia that was successfully treated with phages as adjunct to antibiotics [33]. The initial four-week therapy comprised several antibiotic compounds. After that, the patient relied on supplemental oxygen through a nasal cannula with a flow rate of 6 L min⁻¹ and was still in poor clinical condition. Since distinct P. aeruginosa strains exhibited sensitivity to colistin only, colistin inhalation was initiated. One week later, the patient experienced fever and an enhanced purulent sputum production, which prompted a reinstatement of intravenous antibiotics. In spite of this, the patient's clinical condition deteriorated within the course of the subsequent week as indicated by an enhanced oxygen requirement of 70 L min⁻¹. Then, the phage AB-PA01 was given every six hours for a duration of eight weeks, with the respective intravenous syringe containing 4 × 10⁹ PFU in 5 mL. At the beginning, the patient was supplied with oxygen through a heated high-flow blender at a rate of 30 L min⁻¹. Colistin was withdrawn, but ciprofloxacin and piperacillin were concurrently deployed for three weeks. By the seventh day of phage therapy, the patient was afebrile, but experienced a dry cough and difficulties with sputum's expectoration. At completion, the patient required only 3 L min⁻¹ of supplemental oxygen via the nasal cannula and had no sputum production. Within 100 days after the end of the phage therapy, neither a relapse of P. aeruginosa pneumonia nor a CF exacerbation occurred. Neither clinical nor laboratory monitoring revealed adverse reactions to phage therapy, during which the isolates essentially continued to be sensitive, except for one isolate on day 9 showing transient resistance [33].

Aslam et al. reported the use of phages as a treatment for MDR infections in three cases of lung transplant recipients, two of whom were infected with P. aeruginosa [34]. The first patient, a 67-year-old man with transplanted lungs following hypersensitivity pneumonitis, was administered the phage AB-PA01, both intravenously and via the airway (exemplarily illustrated in Fig. 3) in combination with systemic antibiotics for two weeks, at the end of which the inflammatory conditions were substantially reduced. While the AB-PA01 application via nebulizer was continued for another week and tentatively terminated on day 29, antibiotics were discontinued on day 18. Due to a worsened condition on day 46, antibiotics were reinstated and supplemented by an addition of a Navy phage cocktail 1 and the use of a phage combination, designated AB-PA01-m1, of the primarily used phage cocktail and a new specific phage. The MDR P. aeruginosa pneumonia subsided, and a suppression was attained through Navy phage cocktails 1 and 2 from days 93 up to 150 leading to inactivity of P. aeruginosa for that time and the subsequent three months. The other patient, a 57-year-old female lung transplant recipient with non-CF bronchiectasis, was infected with a MDR P. aeruginosa strain resistant to all antibiotics but colistin. Her treatment involved the intravenous application of AB-PA01 and colistin via the airways for four weeks. While no notable alteration of inflammatory signs was observed in the course of the therapy, no further P. aeruginosa bacteria could be isolated (from the beginning of phage therapy and until 60 days following its end) and an increase of sensitivity to antibiotics was noted at day 60. Neither patient showed adverse effects attributable to the applied phages, but in case of the patient 1, the appearance of phage-resistant isolates was detected after completion of AB-PA01 and AB-PA01-m1, each [34].

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Phage therapy in humans and ways of administration. Phages can reach the pulmonary site of infection through inhalation and systemically following intravenous administration. Hence, the P. aeruginosa bacteria locally abundant in mucous environment are effectively eliminated, facilitating the therapeutic success

Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00060

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Chen et al. delineated a case of bacteriophage therapy of pneumonia and empyema associated with broncho-pleural fistula in a 68-year-old Chinese patient being infected with carbapenem-resistant P. aeruginosa [35]. At first, the authors detected P. aeruginosa in sputum, BAL fluid, pleural effusion, and lung biopsy samples, whereupon the patient was administered antibiotic therapy. After carbapenem-resistant P. aeruginosa was cultivated from pleural effusion, a phage therapy (PA3, PA18) was concomitantly applied. Phage application was performed twice a day by nebulization and, once every day, by injection into the pleural cavity, while antibiotics were given intravenously. On the eleventh and twelfth day of phage therapy, administration of gaseous phages was performed three times, given that P. aeruginosa persisted in pleural effusion days earlier. During the course of phage therapy, a significant reduction in inflammatory signs was observed. Eventually, antibiotic therapy was terminated four weeks after phage therapy and the patient was discharged from the hospital in stable condition. No adverse effects attributable to phage therapy occurred [35].

Key findings

Results from several investigations showed that the utilization of phages, some of which are summarized in Fig. 4, caused a reduction in pulmonal bacterial load [13, 14, 17, 18, 25] and the clearance of P. aeruginosa from the blood stream [14, 18, 31]. The administration of phages was responsible for a milder course of the underlying lung disease and significantly increased the survival of mice [20, 26, 30]. Moreover, phage therapy was associated with a mitigation of P. aeruginosa induced pulmonal damage [13, 14, 16] and attenuated pro-inflammatory immune responses in the host [15–18]. The mice did not exhibit phage-induced adverse clinical signs [31], and if at all, only negligible immunopathological tissue changes were noticed in vitro and in vivo in the presence of phages [13, 16].

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Selected compilation of addressed phages. Terms coined for phages used in the various studies are collocated here. For further details related to the individual phages refer to the main text

Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00060

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The lysis of MDR P. aeruginosa was explicitly achieved by the applied phages [14, 27] and occurred in a dose-dependent manner [14, 18]. Interestingly, the phage titers measured in mice that had been challenged with the lowest dose were eventually comparable to those of mice initially receiving higher doses, whereas doses beyond were associated with an increased risk of resistance development against the phages [18]. Following application, the phage titers increased due to lytic replication [13, 15] and decreased when either therapy was concluded [16] or no bacteria were abundant any more [15]. Although one study reported hepatic phage proliferation [15], no decrease of bacterial loads in the liver was found in another study [18].

The reviewed in vitro phages studies were performed under different experimental conditions, mainly with varying temperatures, pH values, and salt concentrations, but revealed a relatively robust stability of the phages and resistance against adverse external conditions [17, 20]. Moreover, a delayed response to the phage therapy applied to mice did not necessarily result in treatment failure [31]. Repeatedly, phages acting specifically against P. aeruginosa exhibited the beneficial property of wide host ranges for various strains [16, 20, 24]. While no adverse phage-phage interactions were described [19], cross activities of phages from diverse origins were observed [24].

Even phage-derived endolysins alone succeeded in killing P. aeruginosa in vitro and improved the survival of infected mice upon treatment. Similar to phages, the antibacterial efficacy of the endolysins was shown to be dose-dependent and effective against a plethora of P. aeruginosa strains as well as biofilms [21–23]. Some lysins maintained their activity at serum concentrations of 6%–8%, whereas others ceased it at less than 1% [22, 23]. Whereas neither surfactants nor wide ranges of NaCl and proton concentrations counteracted the effects exerted by the lysins [21–23], lysins did neither affect HL-60 neutrophils nor red blood cells [23].

Interestingly, developing resistance to phages rendered MDR P. aeruginosa more sensitive to antibiotics again in several studies [15, 18, 32]. Basically, the respective sensitivity to phages and antibiotics is variable and reciprocally correlated [27]. Therefore, a better outcome was assumed and shown concerning, among others, biofilm degradation when phages and antibiotics were concomitantly deployed [17, 24, 26]. Despite the fundamental ability of phages to penetrate biofilms and kill underlying P. aeruginosa [25], one experiment with a combination of phages and antibiotics encompassed the resurgence of bacterial growth involving new mucoid morphotypes and augmented biofilm formation [24].

While one study negated phage induced immunological reactions [30], the murine immune system responded to phage application with enhanced TNF-α and IL-6 levels [15] as well as phage-specific IgG and CD4⁺ T cells in another report [29]. However, it was asserted that alongside phages, the immune system incorporates the complement system [31] and neutrophilic granulocytes in the joint combat of P. aeruginosa infections. In consequence, phage therapy was discouraged in patients with neutropenia [30], even though, therapeutic success was achieved in neutropenic mice [13, 15, 18]. Interestingly, resistance to phages was accompanied by facilitated elimination through cathelicidin LL-37, underscoring distinct synergistic effects [32] as visualized by Figure. 5.

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Killing of multidrug-resistant (MDR) P. aeruginosa as concerted action of phages, synthetic antibiotics, and the host immune system. Depicted from left to right in the section of the immune system are a neutrophil granulocyte, cathelicidin LL-37, and complements. The structural diagram and the stick model in the antibiotics section represent meropenem and tobramycin, respectively

Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00060

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Case reports of phage therapy against human lung infections caused by MDR P. aeruginosa covered intravenous and inhalational phage administration. Since antibiotics alone failed to cure infected patients, phage cocktails were concomitantly applied [33–35]. As a result, mitigated pro-inflammatory host responses could be observed [34, 35]. On the other hand, the occurrence of cytokines, IgG, CD4⁺ T cells, and neutralizing antibodies were attributed to host immune responses upon the phage therapy itself, further supporting the clearance of the infectious pathogens [28]. All phage treated patients recovered from severe pulmonary infections, whereas virtually no side effects of the phage treatment could be assessed [33–35]. After discontinuation of the phage treatment, no relapses by P. aeruginosa infection were observed. During treatment with a mix of phages, multiple P. aeruginosa isolates may be attacked at a time. Furthermore, the sensitivity of the attacked P. aeruginosa isolate against distinct phage components might shift over time with the development of distinct resistant pathogenic isolates but still resulting in overall clinical resolution [33, 34]. These results further underscore rather the application of a combination of phages instead of single ones in the combat of infections with P. aeruginosa infections, particularly when caused by MDR isolates.

Limitations

Although the thoroughly performed search and selection of publications yielded 23 relevant results, errors and inaccuracies cannot be entirely ruled out. The omission of further publications is also possible given that neither another search engine than PubMed nor its Medical Subject Headings were used. The restricted literature base could possibly have been expanded by utilizing more synonyms (e. g., “extensively drug-resistant”, “carbapenem-resistant P. aeruginosa”, or “lower respiratory tract infection”), for instance.

The comparability of the results is hampered by the pronounced heterogeneity of the studies regarding objectives and methodology. Thus, there are substantial differences in the way phages and phage-derived lysins were applied (e. g., dose, administration form) as well as in the concomitant experimental conditions (e. g., combination of multiple phages or with antibiotics), for instance. These facts entail a clouded significance of this review.

After all, 14 papers refer at least partially to in vivo experiments and only five exclusively to in vitro experiments, whereas three case reports and one brief report refer to a total of as little as five infected patients subjected to phage application. Given this quantitative and qualitative basis, only a rather low level of evidence is conceded and conclusive statements regarding the therapeutic effects and safety of phage treatment in P. aeruginosa infected patients should be undertaken with caution.

Conclusions

In summary, our review revealed that phages and phage-derived endolysins demonstrate favorable features that suggest their suitability for the treatment and prophylaxis of lung infections caused by P. aeruginosa including MDR isolates. In particular, the phage-derived measures have been shown effective against both, MDR P. aeruginosa strains and biofilms without provoking major side effects, except for (subtle) host immune responses. However, both, in vitro and in vivo studies providing deeper insights into the interplay of phages, infectious pathogens, and host immunity are needed. This also applies to prospective placebo-controlled intervention studies in order to further elucidate efficacy and safety of phage treatment and/or prophylaxis of MDR bacterial infections in immune-competent and maybe also immune-compromised individuals.