Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5400_Библиотеки_им_академика_М_И_Перельмана
.pdf
Fig.1 Schematic of the replication of bacteriophages. Bacteriophages can replicate by
either the lytic or the lysogenic cycle depending on their species. Lytic cycle: (1) Phages
recognise their speciic host and dock themselves to the bacterial membrane. (2) The
attached phage releases its DNA into bacterial cells. (3) Phage DNA replicates and
circularises but remains separate from the host DNA. (4) Phage proteins are made from the
replicated DNA. (5) Phages parts assemble. (6) As phages ill the host cell, it lyses thereby
releasing the phages. The lysogenic cycle takes a slightly different course from the lytic
cycle. (A) Once the phage releases its DNA into the bacterial cell, the phage DNA is
integrated into the host genome. The genetic material of phages is referred to as prophages.
(B) Bacterial cell divides, replicating the prophage. (C) The host cells continue to replicate
with the integrated prophage. When the bacterial cell experiences stress, it can excise the
prophage (Step 3) and undergo the lytic cycle to replicate phages
2 InhaledPhagesAgainstMDRPathogens
Phages have demonstrated positive outcomes in both animal and clinical
studies. Therapeutically, lytic phages are preferred as the cycle of infection,
replication, and lysis is quicker than that of lysogenic phages. Its speciicity
and ability to multiply are desirable as it may lower the risk of adverse events
and is able to provide a constant dose supply, meaning it may penetrate
bioilms [15]. Recent successful case studies on treating clinical MDR
infections are promising [12, 13]. Given the rising AMR and lack of new
antibiotic development, it is worth pursuing and realising the therapeutic
potential of phages. Inhaled phage delivery would be more eficient than
other routes of administration for respiratory infections because it targets
pathogens in the lungs directly, elicits faster action, and is non-invasive.
2.1 Staphylococcusaureus
https://t.me/medicina_free
Methicillin-resistant S.aureus (MRSA) commonly causes nosocomial
infections and is resistant to irst-line antibiotics. They are responsible for
ventilator-associated pneumonia (VAP), hence treatment with a phage
cocktail has been investigated [16, 17]. Phages 2003, 2002, 3A, and K at a total
concentration of 1.5
× 1010 plaque forming units (PFU)/mL were nebulised
(vibrating mesh) at 2, 12, 24, 48, 72
hours post-inoculation of the MRSA
mouse model and compared with intravenous (IV) delivery of the phage
cocktail and IV linezolid [16]. The phage cocktail alone was successful in
improving the survival of the mice by 50% compared to control (0%) and IV
linezolid (38%) and was solely localised in the lungs, indicating no
association with systemic side effects [16]. However, when administered
together with an IV phage cocktail, the survival rate dramatically improved to
91% [16]. The nebulised phages only rescued 50% of the mice, possibly due
to poor penetration into the deep lung parenchyma as well as the infection
spreading into the spleen, indicating bacteraemia [16]. Phage therapy could
also be utilised as prophylaxis for VAP [17]. A single high dose of the
nebulised phage cocktail (2003, 2002, 3A, K) 6 hours prior to infection
resulted in 60% survival for 3
× 1010 PFU and 70% for 3 × 1011 PFU compared
to untreated mice, none of which survived [17]. The bacterial load in the lungs
of the surviving mice was approximately 500 times lower than that in the
untreated and dead mice [17]. The authors concluded that these studies could
be the foundations for future clinical trials as currently there are no
randomised controlled trials (RCT) examining inhaled phage therapy against
S.
aureus.
2.2 Klebs iellapneumoniae
K.pneumoniae is an opportunistic pathogen commonly found to cause
community- and hospital-acquired pneumonia. Like many other pathogenic
species, many strains are becoming increasingly multi-drug resistant,
including against carbapenems [18]. Intranasal administration of phages
pKp11 and pKp383 resulted in 100% survival and boasted a greater
eficiency in suppressing the bacterial load in mice infected with a clinical
isolate, K.pneumoniae SY1 compared to treatment with either pKp11 or
pKp383 alone [19]. In a mouse pneumonia model, a single intranasal
administration of monophage 2 hours post-inoculation of K.pneumonia
resulted in 80% survival compared to the PBS control [20]. The bacterial load
detected in phage-treated mice with sublethal pneumonia was 2-logs lower,
the observed lung consolidation was localised, and lung tissues were
predominantly healthy compared to the control. Similarly, another study
which investigated the mouse pneumonia model observed no inlammatory
https://t.me/medicina_free
cell exudate in the bronchioles, focussed areas of necrosis as opposed to
diffuse areas in histopathological lesions, and no perivascular/bronchial
cufing in mice treated with phages compared to control [21]. Overall, there
was a signiicant drop in bacterial load 48 hours post-infection and a
complete elimination was achieved at 96 hours post-infection.
2.3 Ac inetobacterbaumanni
A.baumannii is also another opportunistic pathogen that is often associated
with hospital-acquired pneumonia and carbapenem-resistant A.baumannii
(CRAB) is a growing public health problem. An in vivo study demonstrated
better survival for the intranasal PBAB phage cocktail-treated (PBAB08,
PBAB25) mice compared to the control at day 4 post-infection (60% vs 20%
survival) [8]. A 2-log reduction in bacterial load in the infected lungs of mice
was noted on days 3–4 post-infection. However, a complete elimination of the
bacteria was not achieved. Similarly, treatment with intranasal antibaumannii phage Bϕ-R2096 resulted in improved survival 16 and 48 hours
post-infection compared to control in mice [10]. The authors also reported
that the phage-only treatment did not show any tissue damage and no
serious adverse events were noted. Interestingly, Bϕ-R2096 was able to
completely clear the CRAB infection in the lungs, unlike the PBAB cocktail.
Although this indicates the therapeutic eficacy of phages, it also highlights
the complexity of selecting appropriate phages for therapeutic use. The
differing extent of survival rates could depend on several factors, such as
different strains of CRAB, different mouse types, and dosing regimens.
2.4 Pseudomonasaeruginosa
P.aeruginosa frequently infects cystic ibrosis patients and causes
pneumonia. Mouse inhalation model that realistically relects chronic lung
infection caused by P.aeruginosa demonstrated a 3-log reduction in bacteria
colony forming units (CFU) 24 hours post-intranasal phage PELP20
treatment [11]. Ultimately, a complete clearance of infection was observed in
70% of the mice and the remaining 30% had signiicant CFU reduction 6 days
post-infection [11]. Nebulised phage cocktail therapy rapidly controlled
ventilation-associated pneumonia in a recent porcine model that closely
resembled focal pneumonia in humans [22]. The rapid 1.5 log reduction in
bacterial load may be due to the higher-than-expected phage titre of 107–
10
9
PFU/mL that was observed in the broncoalveolar lavage (BAL) and in
consolidated pneumonia. This suggests that phages can replicate on-site and
penetrate deep areas of infection where there may be limited air passage.
https://t.me/medicina_free
Furthermore, no phages were detected in the serum or urine, again
highlighting its localised action.
Success of phage therapy against P.aeruginosa was also observed in
humans, but only through intravenous administration. A case study of a cystic
ibrosis patient with pneumonia caused by MDR P.aeruginosa was
administered with an intravenous (IV) cocktail of four lytic phages at a dose
of 4
× 109 PFU/5 mL every 6 hours for 8 weeks [13]. This patient remained on
azithromycin and piperacillin-tazobactam prior to and throughout the phage
course and had intermittent use of cipro
loxacin and doripenem during phage
therapy. By the end of the phage course, the infection was fully resolved
without adverse reactions and with no recurrence within 100 days. Another
case study demonstrated the success and safety of phage monotherapy in a
lung transplant patient with P.aeruginosa pneumonia [12]. However, these
are isolated success reports in which phages were used under compassionate
grounds. They only serve as non-systematic evidence to encourage further
clinical trials. Furthermore, phage therapy in humans has been limited to oral,
IV, and topical routes. The inhalation route is yet to be investigated, despite
the countless number of studies that have reported its eficacy. Currently,
there are three registered RCTs that evaluate the safety and/or eficacy of
inhaled phages against P.aeruginosa (clinical trial identiier: NCT05616221,
NCT04596319, NCT05010577) [23–25]. The scarcity of clinical evidence on
inhaled phages may be due to the lack of robust safety data and hesitation in
administering phages that were not produced by Good Manufacturing
Practice (GMP). The PhagoBurn trial in 2019 [26] was the only double-blind
RCT investigating the eficacy of phages that were produced by GMP and
gained approval from health regulatory agencies in France, Belgium, and
Switzerland.
2.5 Mycobacteriumtuberculosis
M.tuberculosis is an intracellular bacterium that causes tuberculosis and can
quickly mutate to become resistant to antibiotics and typically targets lung
alveolar macrophages [27]. As there are numerous natural phages, there
would be ones that speciically target M.tuberculosis. An example is phage
D29, which was used as prophylaxis against the pathogen [28]. A dose of 1
plaque forming unit (PFU)/alveolus was delivered using nose-only inhalation
to mice via a vibrating mesh nebuliser 30 minutes prior to M.tuberculosis
H37Rv infection [28]. Prophylaxis treatment with nebulised D29
demonstrated a signiicant reduction in bacterial load 24 hours postinfection (approximately one-third of the untreated mice) and continuously
maintained a low bacterial burden 3 weeks post-infection [28]. Although the
https://t.me/medicina_free
infections were not caused by M.tuberculosis, phage therapy has been
compassionately used to treat 20 patients against infections caused by the
non-tuberculosis-causing Mycobacterium spp. [29]. The outcome was
inconclusive, with one patient completely resolved of infection after 1 year of
IV phage treatment, whilst one patient stopped treatment after 6 months due
to a lack of response [29]. Another patient was also completely cleared of the
infection, but only after receiving IV phages for 10 months plus two direct
bronchoscopic administrations. One patient was free from infection after IV
treatment for 1 year but is still continuing therapy [29]. Nineteen out of
twenty patients initially received IV phage therapy. Five of them changed to
aerosolised phage treatment that is still ongoing at the time of writing [29].
The authors did not specify how they aerosolised the phages. Treatment of
Mycobacterium is dificult as phages would need to enter, and remain viable
after entering, the host cell in order to infect the bacteria inside. Although
clinical evidence is limited so far, utilising phages to treat intracellular
bacterial infections is promising. More research in this area is needed.
3 PhageSynergy
Phages provide a promising treatment against AMR as some phages have
synergy with antibiotics. The synergy manifests through enhancing the
susceptibility of the bacteria to antibiotics, increasing the lytic activity of the
phages, or a higher reduction of the bacterial load. It may be possible to delay
or even avoid the development of phage-resistant bacteria [30]. The task
force convened by the Antibacterial Resistance Leadership Group
recommended that phage therapy should be used in conjunction with
conventional antibiotics [14], increasing the need to investigate the
synergistic effects of phages and antibiotics. Studies have shown that synergy
depends on the species of the phage.
Ciproloxacin is often used to treat respiratory infections and works
synergistically with phages. Lin et al. [31] reported that anti-Pseudomonas
phage PEV20 and ciproloxacin displayed a synergistic effect which was
maintained even after jet nebulisation. The mechanism behind the synergy
was suggested to be due to PEV20 changing the activity of the eflux pump on
the bacteria to increase the intracellular concentration of ciproloxacin which
then inhibits bacterial DNA replication [31] (Fig. 2). Lin et al. [32] also
demonstrated that the synergy was maintained in a mouse lung infection
model when co-spray dried PEV20 with ciproloxacin was delivered
intratracheally, which reduced CD8+T cells, B cells, and lung
monocytes/macrophages. However, the decreased in
lammatory response
https://t.me/medicina_free
might be due to the rapid control of bacterial infection by ciproloxacinPEV20 and not necessarily due to active anti-inlammation by the
combination. Other P.aeruginosa targeting phages showed synergy with
antibiotics by reducing antibiotic resistance but it should be noted that it was
not delivered via the respiratory route [30, 33–35]. In fact, the two studies by
Lin et al. [31, 32] are the only published studies so far that examine inhaled
phage antibiotic synergy (PAS) [36]. The same phage cocktail as that used in
PhagoBurn (PP1131) reduced the bacterial load of the resistant P.aeruginosa
CHA strain in an in vitro ibrin-clot model 6 hours post-administration, but
not the P7 bacterial strain [30]. However, bacterial regrowth was observed
beyond 6 hours post-administration, indicating phage-resistance
development. In vivo, endocarditis was induced in rats by inoculation with P.
aeruginosa CHA and treated with phage and ciproloxacin intravenously. The
combination resulted in negative vegetation cultures in 7 out of 11 rats
6 hours after treatment whereas ciproloxacin-alone and phage-alone had no
rats with negative cultures. Unlike in vitro, phage resistance was not detected
in vivo where co-therapy with ciproloxacin was applied [30]. Phage
resistance in vivo is not commonly observed due to a itness trade-off or
clearance by the immune system [37, 38], which may be the case in the study
by Oechslin et al. as neutropenic mice were not used. Further investigation
has identiied the characteristics of phage-resistant bacterial strains. The P.
aeruginosa 24/2 strain had a disrupted pilT gene which produces ATPase for
type IV pilus contraction, whereas the P.aeruginosa 19/2 strain had a
truncated lipopolysaccharide (LPS) that resulted in reduced virulence. Type
IV pili and LPS are also phage receptors in which disruptions have resulted in
phage resistance and hence the regrowth of P.aeruginosa 6 hours post-
treatment [30]. The regrowth of P.aeruginosa in the presence of a cocktail of
12 different phages implied that the therapeutic dose of the individual phages
was suboptimal, resulting in ineffective clearance of the bacteria [30]. It could
also be due to the various phages not complementing each other [30].
Cocktails should be designed strategically, considering the host range and
targeting receptors on the bacteria to be effective in reducing or eliminating
phage-resistance development [39].
https://t.me/medicina_free
Fig.2 Proposed mechanism of action of phage-antibiotic synergy. (A) Lin et al. [31] have
suggested that phage PEV20 may induce changes in the eflux pumps of P.aeruginosa,
increasing the intracellular concentration of ciproloxacin (represented by pink pentagons).
Further clariication into how PEV20 may interact with the eflux pumps has not been
explored. (B) Gurney et al. [33] proposed that phage OMKO1 may bind to the surface of P.
aeruginosa via Type-IV pili, causing the pili to retract for an easier interaction with the
OprM protein of the eflux pump. Ultimately this will retain ciproloxacin concentration in
the bacteria. This mechanism is also applicable to tetracycline. (C) The high intracellular
concentration of ciproloxacin works to inhibit DNA gyrase and topoisomerase IV to disrupt
DNA synthesis, leading to cell death
Similarly, co-culturing P.aeruginosa with phage OMKO1 resulted in a >60%
reduction in antibiotic resistance to erythromycin, tetracycline, and
ciproloxacin and also displayed “phage steering” where phage-resistance is
traded off with antibiotic-resistance [33]. OMKO1 increased bacterial
susceptibility to ciproloxacin and tetracycline in addition to preventing
antibiotic resistance evolution (Fig. 2). Ceftazidime was also used with
OMKO1 to treat prosthetic vascular graft infections in a patient [34]. The fact
that phage OMKO1 works synergistically with anti-pneumonia drugs, such as
ciproloxacin and ceftazidime, makes it a good candidate for the respiratory
route. Direct delivery of OMKO1 and either of these antibiotics to the lung
may exert a more eficient response coupled with PAS to quickly subdue the
https://t.me/medicina_free
bacterial infection. This may also be applicable to the phage cocktail PP1131.
The exploration of the respiratory route with PP1131 would be a big step in
inhaled phage therapy as the phage cocktail was manufactured by GMP.
Furthermore, Roach et al. [38] showed that the immune system plays a major
role in delivering effective inhaled phage therapy. Phage therapy is
multidimensional. The literature indicates that phages could be used to
potentiate antibiotics to once again target bacterial infections which were
once multi-drug resistant.
4 ImmuneResponsesInducedbyRespiratoryDeliveredPhages
Cytokine storms can be observed in both infectious and non-infectious
diseases and can also result from therapeutic interventions [40, 41]. In the
event of a cytokine storm during severe lung infections, local inlammation
overlows into the systemic circulation and causes sepsis [40]. As the
therapeutic phages are lytic, the possibility of an uncontrolled surge in
endotoxin release following bacterial lysis and the consequent proinlammatory response cannot be dismissed. Even when phages do not cause
a cytokine storm, they may elevate, suppress, or modulate both proinlammatory and anti-inlammatory cytokines. To fully utilise the
therapeutic potential of phages, we must investigate the nature of the
immune responses that phages induce in vivo.
There is currently a limited number of studies on the immune response of
phages targeting P.aeruginosa via the respiratory route and their results are
inconclusive (Table 1). Intranasal delivery of a phage cocktail targeting P.
aeruginosa reduced the bacterial load and lowered pro-inlammatory
cytokines, including IL-6, TNF-α, keratinocyte chemoattractant, and IL-12p70
in mice [42]. Similarly, in the treatment of pneumonia colonised with K.
pneumoniae, intranasally delivered phage 1513 signiicantly reduced TNF-α
and IL-6 levels than the untreated mice [20]. However, the changes in the
immune response were only observed in the presence of the bacteria so it is
unclear whether the cytokine reduction was due to phage activity alone or
due to the bacterial load reduction.
Table1 Immunological effects of respiratory delivered phages in animal lung infection
models
https://t.me/medicina_free
Phage Pathogen Routeof
administration/animal
Dose Immunological
effects
Reference
Phage
cocktail 1
(contains:
24, 25, 7)
P.
aeruginosa
PAO1
Intranasal/BALB/c mice
1.24
× 10
9
PFU
Reduced IL-10,
IL-6, TNF-α,
keratinocyte
chemoattractant,
IL-12p70 in the
bronchoalveolar
lavage
luid
Phage treatment
signiicantly
reduced
neutrophils
compared to
untreated mice
[42]
KP 1513 K.
pneumoniae
KP 1513
Intranasal/SwissWebster mice
2
× 10
9
PFU
Reduced IL-6
and TNF-α in
lung tissue
compared to
untreated mice
[20]
536_P1,
LM33_P1
E.
coli 536,
E.coli LM33
Intranasal/BALB/c mice
107 CFU
for phage
536
5
× 10
7
CFU for
phage
LM33
No change in
complete blood
count
LM33_P1
reduced
lymphocyte
count in the
blood
536_P1 elevated
IFN-γ, IL-12,
MCP-1 and
keratinocyte
chemoattractant
in the lung at
17
hours postadministration
in lungs but not
in the blood
[43]
Pf4 P.
aeruginosa
PAO1
Intratracheal/C57BL/6
mice
1.5
× 10
7
PFU
Reduced CXCL1,
IL-17, IL-1β, IL17 and IL-10 in
the
[44]
https://t.me/medicina_free
Phage Pathogen Routeof
administration/animal
Dose Immunological
effects
Reference
bronchoalveolar
lavage luid
IL-12p40 in the
bronchoalveolar
lavage luid was
signiicantly
higher at
48 hours postadministration
compared to
PAO1-infection
alone
No signiicant
differences in
the number of
macrophages
after phage
treatment
Phage
cocktail
(contains:
PBAB08,
PBAB25,
PBAB68,
PBAB93)
A.baumanii
(14 clinical
strains)
Intraperitoneal,
intranasal, oral/BALB/c
mice
109 PFU
Minimal
increase in
serum cytokine
(IL-1a, IL-1b, IL2, IL-4, IL-6, IL10, IL-12, IL17A, TNF-a, GCSF) in all
routes.
Increased GMCSF in all routes
of
administration
[8]
PAK_P1 P.
aeruginosa
PAKlumi
Intranasal/BALB/c,
C57B/6/J, Rag2
−/
−
Il2rg
−/−
, Myd88
−/−
mice
108 PFU
No signiicant
increase in
cytokine
production in
lung tissue
except IFN-y and
TNF-α
Synergy
observed
between the
phage and the
[38]
https://t.me/medicina_free
Соседние файлы в папке Библиотека им академика М.И. Перельмана