Case report: Local bacteriophage therapy for fracture-related infection with polymicrobial multi-resistant bacteria: hydrogel application and postoperative phage analysis through metagenomic sequencing

Frontiers in Medicine 01 frontiersin.org

Case report: Local bacteriophage

therapy for fracture-related

infection with polymicrobial

multi-resistant bacteria: hydrogel

application and postoperative

phage analysis through

metagenomic sequencing

VolkerAlt

1†

, AndréGessner

2†

, MayaMerabishvili

FlorianHitzenbichler

, GopalaKrishnaMannala

DavidPeterho

, NikeWalter

, Jean-PaulPirnay

AndreasHiergeist

2‡

and MarkusRupp

‡

Department of Trauma Surgery, University Hospital Regensburg, Regensburg, Germany,

Institute of

Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg, Germany,

Laboratory for Molecular and Cellular Technology (LabMCT), Queen Astrid Military Hospital, Brussels,

Belgium,

Department of Infection Prevention and Infectious Diseases, University Hospital

Regensburg, Regensburg, Germany

Fracture-related infections can be challenging, particularly with concomitant

severe bone defects and multi-resistant microorganisms. We present a case

of a 42-year-old patient with a fracture-related infection following a war

injury from a gunshot, resulting in a 12-cm subtrochanteric segmental bone

defect and the detection of four dierent multi-resistant Gram-negative

bacteria. Due to antibiotic drug resistance, treatment with bacteriophages was

considered. Phage susceptibility testing revealed the activity of a commercially

available bacteriophage cocktail (Intesti bacteriophage, Eliava Institute, Tbilisi,

Georgia). This phage cocktail was included in a modiﬁed two-stage Masquelet

technique. During the ﬁrst intervention, the bone was debrided and samples for

microbiological and phage testing were harvested. The indwelling intramedullary

rod was removed, and the bone defect was ﬁlled with a PMMA spacer loaded

with colistin and the bone stabilized with a plate. During the second procedure,

the PMMA spacer was removed and a silver-coated angular stable plate was

implanted. The bone defect was ﬁlled with a ﬁbular autograft and allograft

cancellous bone chips. At the end of the procedure, the Intesti bacteriophage

cocktail was injected into a DAC hydrogel and this bacteriophage hydrogel

composite was then put onto the angular stable plate. Postoperatively the wound

ﬂuid was collected over 72 h, and high-throughput metagenomic sequencing

was performed. This showed a time-dependent release of the bacteriophages

in the wound ﬂuid, with a relatively high concentration after 12 h, decreasing to

DNA copies of 0 after 72 h. Furthermore, wehave assessed the release of phages

from DAC gel and the eect of DAC gel on the phages in vitro. The results

showed a stable and rapid release of phages from the DAC gel (~1×10

PFU/mL).

The clinical course of the patient showed no relapse of the infection with good

bone consolidation of the bone defect after 1 year without the need for any

surgical revision. To the best of our knowledge, this is the ﬁrst case that shows

the detection of bacteriophage DNA copies by high-throughput metagenomics

OPEN ACCESS

EDITED BY

William Calero-Cáceres,

Technical University of Ambato, Ecuador

REVIEWED BY

Seth Commichaux,

UnitedStates Food and Drug Administration,

UnitedStates

Swapnil Ganesh Sanmukh,

Université Clermont Auvergne, France

*CORRESPONDENCE

Markus Rupp

markus.rupp@ukr.de

†

These authors share ﬁrst authorship

‡

These authors share senior authorship

RECEIVED 06 May 2024

ACCEPTED 20 June 2024

PUBLISHED 12 July 2024

CITATION

Alt V, Gessner A, Merabishvili M,

Hitzenbichler F, Mannala GK, Peterho D,

Walter N, Pirnay J-P, Hiergeist A and

Rupp M (2024) Case report: Local

bacteriophage therapy for fracture-related

infection with polymicrobial multi-resistant

bacteria: hydrogel application and

postoperative phage analysis through

metagenomic sequencing.

Front. Med. 11:1428432.

doi: 10.3389/fmed.2024.1428432

Hitzenbichler, Mannala, Peterho, Walter,

Pirnay, Hiergeist and Rupp. This is an

open-access article distributed under the

terms of the Creative Commons Attribution

License (CC BY). The use, distribution or

reproduction in other forums is permitted,

provided the original author(s) and the

original publication in this journal is cited, in

accordance with accepted academic

practice. No use, distribution or reproduction

is permitted which does not comply with

these terms.

TYPE Case Report

PUBLISHED 12 July 2024

DOI 10.3389/fmed.2024.1428432

Alt et al. 10.3389/fmed.2024.1428432

Frontiers in Medicine 02 frontiersin.org

sequencing in a patient with a complex fracture-related infection. Successful

treatment of this case encourages further investigation of bacteriophage

therapy in patients with complex bone and joint infections.

KEYWORDS

bacteriophage, fracture-related infection, metagenomic, Masquelet, hydrogel

1 Introduction

e treatment of fracture-related infections (FRI) is challenging.

Particularly, the involvement of severe bone defects and multi-

resistant bacteria frequently leads to complex situations. e treatment

of FRIs mainly consists of two key aspects: First, control of the

infection and causative microorganisms must beachieved. Second,

bone reconstruction with subsequent successful bone healing needs

to beobtained (1).

Both local and systemic antibiotic therapy are hallmarks of

fracture-related infection treatment. However, there are oen only

limited antibiotic treatment options when multi-drug resistant

bacteria are involved. An alternative or complementary approach for

the treatment of complex infections with multi-drug resistant bacteria

is bacteriophages (short phages). e bactericidal eect of phages was

rst identied more than 100 years ago by Felix d’Herelle (2). Since

then, phages have been studied and used mainly in the former Soviet

Union during the second half of the 20th century. In recent years,

bacteriophage therapy has also gained interest in the Western world

due to the rise of multi- and pan-drug-resistant bacteria, including

dicult-to-treat bone and joint infections (3). However, there are only

a few published case series or case reports on the treatment of

musculoskeletal infections with bacteriophages (4). A recent review

identied 33 bone and joint infection cases treated with phages, out of

which notably 29 (87%) achieved either microbiological or clinical

success. Furthermore, 8 of the 33 cases (24%) experienced mild and

temporary adverse eects, but there were no reports of serious

complications (5). In addition, underlining the ecacy of phage

therapy in resolving dicult-to-treat infections, Pirnay etal. have just

recently published clinical outcomes of their rst 100 cases treated

with personalized phage therapy. Bone infections were the three most

common indications. Overall, in 77.2% of targeted infections, patients

experienced clinical improvement. In 61.3% of infections where

relevant bacteriological follow-up data were available, eradication of

the targeted bacteria was observed (6). Ongoing clinical trials aim to

rene phage application mode and advance our understanding of how

to eectively manage bone infections. Currently there are a few clinical

trials registered, which already entered the phase of recruiting patients

for phage treatment.

Hitherto, however, the optimal administration of bacteriophages

has not yet been elucidated. Phages can beadministered intravenously

or locally. Dierent approaches with varying quantity, frequency, and

type of application have been described to date (4, 7). Recently, Ferry

et al. published a case report with the use of hydrogel as carrier

material for the application of bacteriophages in a patient with a mega

prosthetic hip infection (8). is study showed a rapid release of the

phages from the DAC hydrogel in an in vitro setting. However, no in

vivo data for the release of the phages in the patient were presented.

erefore, in this study, wepresent a clinical case involving the

loading of bacteriophages into a hydrogel in an FRI. Additionally,

weassess the in vivo release kinetics of the phages from the hydrogel

in the patient’s wound using high-throughput metagenomic

sequencing-based characterization of the administered bacteriophage

cocktail, followed by real-time PCR quantication of selected

bacteriophages in the drainage uid.

2 Case description

A 42-year-old man suered a war injury during the Russian war in

Ukraine. He had a history of a gunshot and a blast injury to his

proximal le femur 8 months ago. Hefurther sustained an open head

injury and a fracture of the 3rd metacarpal of his le hand. e injury

of the proximal femur also included a lesion of the sciatic nerve, mainly

aecting the peroneal part. e patient has been treated in Ukraine

with several debridement and irrigation procedures of the le femur,

along with the application of a ring xator. Multiple revision surgeries

for debridement, and irrigation of local antibiotics resulted in a 12-cm-

wide segmental subtrochanteric bone defect. Aer the transferal to

Germany, the external xator was removed, and the implantation of an

antibiotic-coated intramedullary rod was performed. e

microbiological results of this revision surgery revealed several multi-

drug resistant pathogens such as multi-drug resistant (MDR)

Pseudomonas aeruginosa, MDR Proteus mirabilis, MDR Klebsiella

pneumoniae, MDR Escherichia coli, and vancomycin-resistant

Enterococcus faecium. A combination therapy with daptomycin,

cederocol, and fosfomycin was established. Aer presentation in our

department, a modied two-staged Masquelet procedure was

performed with the removal of the antibiotic-coated rod and

debridement of the subtrochanteric bone defect. Microbiological

analysis of the tissue samples harvested during the rst Masquelet

procedure surgery revealed Cutibacterium acnes, which was sensitive

to penicillin G, vancomycin, and clindamycin. In addition,

S. epidermidis susceptible to vancomycin, rifampin, and cotrimoxazole

was detected. e previously cultivated multi-resistant Gram-negative

bacteria could not bedetected. e 12-cm segmental bone defect was

managed with a daptomycin- and colistin-loaded polymethyl

methacrylate (PMMA) spacer (80 g Copal

(Heraeus Medical,

Germany) + 6 Mio. I.E colistin/CMS and 2 g daptomycin), and proximal

femur was stabilized with an angular stable plate (NCB, Zimmer

Biomet, USA). Systemic antibiotic therapy was initially performed with

fosfomycin 3x5g, colistin (CMS) 3x3MioIU, and daptomycin 1x1g and

administered until nal surgery. Cultures of the initially evidenced

MDR pathogens were sent to the Queen Astrid Military Hospital,

Brussels, Belgium, for phage susceptibility testing. Aer testing the

available phages, the best possible option for additional treatment

Alt et al. 10.3389/fmed.2024.1428432

Frontiers in Medicine 03 frontiersin.org

seemed Intesti bacteriophage cocktail (Eliava Institute, Tbilisi,

Georgia). e phage cocktail then was used with a hydrogel carrier to

improve the release kinetics of this composite biomaterial, as described

in a recently published case by Ferry etal. (8). Aer 92 days during

which phages were tested and wound healing was achieved, the nal

surgery for bone defect reconstruction was performed. e patient was

informed about the intended compassionate use of phages according

to §37 of the Helsinki Declaration, and written consent was obtained

(9). e indwelling PMMA spacer and the angular stable plate were

removed, and the bone ends were debrided again. Five tissue samples

were sent for microbiological analysis, which revealed no detectable

pathogen. Perioperative antibiotic therapy with daptomycin and

meropenem i.v. was supplemented with colistin i.v. for 14 weeks

postoperatively and nished aer negative long-term incubation.

ere was no sign of persistent infection, and the decision was taken

to perform a bone reconstruction with an ipsilateral autologous bula

gra and allogenous cancellous bone within a nicely formed Masquelet

membrane. e subtrochanteric bone region was stabilized by a

combination of a long proximal femur nail (Gamma nail, Stryker,

USA) together with a silver-coated (HyProtect™ coating, Bio-Gate,

Germany) angular stable plate (NCB Zimmer Biomet, USA). e

angular stable plate was slightly bent in order to allow a high volume

of bone graing of the defect with a subsequent spindle-shaped callus

formation. e Defensive Antibacterial Coating (DAC

, Novagenit,

Italy) gel loaded with bacteriophage cocktail was applied onto the plate

before wound closure (Figure1).

Wound drainage uid and serum samples taken aer 12, 24, 48,

and 72 h were evaluated regarding the release of bacteriophages from

the hydrogel into the wound.

e patient was then followed up for 12 months. e wound

healed uneventfully, and there was no sign of reinfection, nor was

there a need for further surgical revision. X-rays and computed

tomography demonstrated sucient bone healing of the graed area,

achieving bone consolidation aer 12 months.

3 Materials and methods

3.1 Testing activity of phages on the patient

strains

e activity of phages was tested by spot-test using 100-fold

dilutions of Intesti bacteriophage cocktail (e Eliava IBMV),

individual phages, phages produced as active pharmaceutical

ingredients (API) available from the collection of Queen Astrid

Military Hospital, Brussels, Belgium. Intesti bacteriophage is a mix

of sterile ltrate of phage lysates active against Shigella spp.

(Shigella exneri serotype 1,2, 3, 4 and Shigella sonnei) (titer no less

than 10

 mL

−1

), Salmonella spp. (S. patarype A, S. paratype B,

S. typhimurium, S. enteritidis, S. cholera suis, S. oranienburg) (titer

no less than 10

mL

−1

), dierent types of E. coli (titer no less than

mL

−1

), Proteus mirabilis and Proteus vulgaris (titer no less than

mL-

), Staphylococcus aureus (titer no less than 10

 mL

−1

Pseudomonas aeruginosa (titer no less than 10

 mL

−1

), and

Enterococcus spp. (titer no less than 10

mL

−1

). Inactive ingredients

are bacterial growth media, standard sodium saline, and

Chinazolin as a preservative. Phage APIs have quality control

certicates from Sciensano (formerly known as the Belgian

Scientic Institute of Public Health) and are ready-to-use

pharmaceutical ingredients to be used in magistral phage

preparations (10). Shortly, each phage was diluted 100 times in

DPBS and 10 μL of each dilution was spotted on the lawn of

bacterial strains made with semi-solid 0.6% lysogenic broth (LB)

agar as a second layer on the surface of LB agar in square Petri

plates. e starting concentration of each phage except Intesti

bacteriophage was 10

plaque-forming units per milliliter (PFU/

mL). e results were recorded aer overnight incubation at

32°C. Individual plaques were counted where visible and eciency

of plating (EOP) was dened as a ratio of the titers of phage on the

test strain and the host strain.

3.2 High-throughput metagenomic

sequencing

In order to analyze the composition of the Intesti

bacteriophage cocktail, metagenomic DNA was extracted from

2 mL of the original suspension before administration. DNA was

extracted following the protocol of Shkoporov etal. (11). Briefly,

viral particles were precipitated by treatment with polyethylene

glycol followed by DNAse and RNAse digestion of free nucleic

acids. DNA was extracted from the precipitate containing

bacteriophages using phenol/chloroform extraction. Sequencing

was performed on the original bacteriophage suspension. For this

purpose, DNA was fragmented to ~150 bp using the M-220

focused ultrasonicator (Covaris, Woburn MA, USA) followed by

two-sided size selection with MagSi-NGSPREP-PLUS beads

(magtivio, Nuth, The Netherlands) using a DNA to bead ratio of

1:1.1 and 1:0.6. The Ion Plus Fragment Library Kit (Thermo

Fisher Scientific, Whatman, MA, USA) was used to prepare the

sequencing libraries. The final library was quantified using the Ion

Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific,

Whatman, MA, USA) and diluted to a concentration of 70

pM. The sequencing library was amplified using the Ion 550™ Kit

on an Ion Chef instrument and loaded onto a 550 chip. High-

throughput sequencing was performed on an Ion GeneStudio™

S5 Plus instrument, resulting in a total of 3.9 million reads with

an average read length of 161 bp.

3.3 Analysis of viral metagenomic

sequencing data

Raw sequencing reads were obtained from the Torrent Suite

v5.18.1 instrument soware and then quality-ltered using

Trimmomatic v0.9 (12). Sequencing adapters were removed using

cutadapt v4.0 (13). Reads mapping to the human GRCh37 reference

genome were removed using bowtie2 v.2.5.0 and samtools v1.17 (14,

15). Filtered reads were assembled using megahit v1.2.9. e VIRify

pipeline was used to detect and classify viral contigs (16). Sourmash

v4.8.0 was used for viral taxonomic classication of viral contigs by

MinHash sketching and searching against the viral genbank 2022.03

database (17). Here, a k-mer length of 31 and a scaled value of 1,000

were used for calculating signatures from assembled viral contigs.

Default parameters were used for taxonomic classication using the

Sourmash tax algorithm.

Alt et al. 10.3389/fmed.2024.1428432

Frontiers in Medicine 04 frontiersin.org

3.4 Quantiﬁcation of bacteriophage DNA in

drainage ﬂuid

In order to quantify viral bacteriophage genomes in wound

drainage uid or blood, a total of 10 mL sample material was taken

at ve dierent time points aer surgery: immediately aer surgery,

12 h post-surgery, and on days 1, 2, and 3 aer surgery. DNA

extraction was executed as described above. e two largest contigs,

which matched to Proteus and Pseudomonas bacteriophage

genomes, were selected to quantify bacteriophage DNA copies from

the original bacteriophage suspension and drainage uids collected

at dierent times: First 12 h, 12 h, and 1, 2, and 3 days post-

operatively. Primers S12_k141_521_f (5’-TATCTATCCCTCTCC

CGCCG-3′) and S12_k141_521_r (5’-GTTGAAGATAACGCCG

ACGC-3′) were used to detect the viral contig VC_521

(Pseudomonas phage KPP10). Primers S12_k141_755_f (5’-AGTGT

GTACAGAGCCAGTGC-3′) and S12_k141_755_r (5’-GCGGTAT

CACCAGCTAGCAT-5′) were designed to target a fragment of viral

contig VC_755 (Proteus phage vB_PmiP_RS1pmA). Fragments

cloned into the pJET 1.2 cloning vector (ermo Fisher Scientic,

Whatman, MA, USA) were used to quantify bacteriophage DNA

from nucleic acid extracts on a LightCycler 480 II using the

LightCycler

480 SYBR Green IMaster (Roche Molecular Sciences,

Rotkreuz, CH).

3.5 The release of pages and DAC gel eect

on the phages in vitro

To analyze the release of the phages from DAC gel and the eect

of the DAC gel on the phages, wehave incubated the DAC gel loaded

with Intesti bacteriophage in PBS at 37°C. e activity of the phages

was tested against the E. coli at dierent time points (0 h, 0.5 h, 1 h, 2 h,

4 h, and 6 h) using double-layer agar method.

4 Results

All ve strains of dierent species were tested against the phage

collection of Queen Astrid Military Hospital, Brussels, Belgium, and

the Intesti bacteriophage cocktail produced by the Eliava IBMV. In

particular, P. aeruginosa strain was tested against 6 phage APIs and 18

individual phages. Only four individual phages proved to beactive

with EOP in the range of 1.0–0.01; three active phages were

representatives of the Pakpunavirus species. e nomenclature

aliation of the fourth phage was not known.

E. faecium strain was tested against 1 API and 4 individual

phages. K. pneumoniae strain was tested against 27 individual

phages. E. coli tested was tested against 5 individual phages. None

of the APIs or individual phages showed any activity on the above

FIGURE1

Subtrochanteric segmental bone defect of the left femur was temporarily stabilized with an intramedullary custom-made PMMA bone cement-coated

rod (A). After removal of the rod, a PMMA spacer with colistin was added for dead space management and the bone stabilized with an angular stable

plate (B). The second stage of the Masquelet procedure was performed with an intramedullary nailing and augmentation plate. During bone defect

reconstruction phages were applied to the DAC® gel (C,D). The DAC gel loaded with Intesti bacteriophage was ﬁnally placed on the silver-coated

plate (E). Postoperative X-ray demonstrates the bone defect ﬁlled with autograft ﬁbula graft and allogenic cancellous bone (F). After 12 months,

sucient bone healing was conﬁrmed by performing X-ray (G) and computed tomography (H).

Alt et al. 10.3389/fmed.2024.1428432

Frontiers in Medicine 05 frontiersin.org

three strains. P. mirabilis was tested only with an Intesti

bacteriophage cocktail. Intesti bacteriophage showed activity

only against the E. coli strain. e titer of the phage(s) in the

cocktail active against the E. coli strain of the patient was dened as

2 ×10

PFU/mL.

As no active phage API was available against the bacterial strains and

the patient needed to betreated urgently, it was decided to start treatment

with Intesti bacteriophage, as the only available option at that time.

4.1 In vivo assessment of bacteriophage

release from hydrogel

In order to characterize the composition of bacteriophage genomes

within the Intesti bacteriophage suspension, nucleic acids were

extracted directly from the administered bacteriophage suspension

using whole shotgun metagenomic sequencing. A total of 511,078 (72,7

Mbp) raw sequencing reads were assembled to 149 contigs

(N50 = 9,914 bp). Of these, 77 could beidentied as viral contigs using

the VIRify pipeline. e taxonomic assignment resulted in 10 dierent

bacteriophage genera, which comprised Seunavirus, Saphexavirus,

Nankokuvirus, Kayvirus, Tunavirus, Hanrivervirus, Chivirus,

Bruynoghevirus, Teseptimavirus, and unclassied Casjensviridae.

Further k-mer-based searching against the GenBank nt database-

matched bacteriophages that target bacterial hosts Staphylococcus,

Enterococcus, Proteus, Shigella, Salmonella, Escherichia coli, and

Pseudomonas (Figure2). is was fully in line with the list of bacterial

hosts in the Intesti bacteriophage package insert.

To measure the release of bacteriophages from hydrogel in

drainage samples, specic real-time quantitative PCR protocols were

developed. ese protocols were based on two selected metagenomic

viral contigs, identied through k-mer-based searching against the

GenBank nt database, which presumably encodes bacteriophages

targeting bacterial species of the genus Proteus and Pseudomonas.

Genomic copies of bacteriophage from the Intesti bacteriophage

cocktail prior to administration and from drainage samples taken at 5

dierent time points aer surgery were quantied by qPCR:

immediately aer surgery, 12 h post-surgery, and on days 1, 2, and 3

aer surgery. e analyses revealed that bacteriophages were released

from the hydrogel within the rst 3 days following surgery (Figure3).

No DNA of these two bacteriophages was detected by qPCR in the

blood samples taken at the same time points.

4.2 In vitro assessment of bacteriophage

release from DAC hydrogel

e release kinetics of the DAC hydrogel injected with Intesti

bacteriophages showed the rapid release of the phages from hydrogel

aer incubation in PBS. ere were no dierences in the release of

bacteriophages with the time progress (0 h-6 h) at 37°C incubation.

e released phage titer was between 1.6 × 10

and 1.9 × 10

PFU/mL,

which indicates a fairly consistent release rate of the phages from the

DAC gel (Figure4).

5 Discussion

In the present case of a Ukrainian soldier wounded in the Russian-

Ukrainian war, a combination of local bacteriophage therapy with

established therapeutic procedures achieved infection eradication and

bone reconstruction of the injured leg despite chronic osteomyelitis

caused by multiple MDR bacteria. In vitro analysis evidenced a

consistent release of phage from the DAC hydrogel. Furthermore,

phage analysis conducted in the postoperative course demonstrated

the activity of phages in the wound area during the rst postoperative

days, with no systemic spread of phages through the blood detectable

FIGURE2

Composition of the Intesti bacteriophage cocktail as revealed by

whole shotgun metagenomic sequencing. Viral contigs were

analyzed by k-mer-based searching against the GenBank nt

database.

FIGURE3

Quantiﬁcation of bacteriophage release by real-time PCR targeting

viral contigs encoding a Proteus and Pseudomonas targeting

bacteriophage in postoperative drainage ﬂuid as well as in Intesti

bacteriophage suspension (viral copies per mL). Samples were

collected at 5 dierent time points: immediately after surgery, 12 h

post-surgery, and on days 1, 2, and 3. The time point of surgery is

marked with a dashed line.

Alt et al. 10.3389/fmed.2024.1428432

Frontiers in Medicine 06 frontiersin.org

within the initial 3 postoperative days. No side eects of phage therapy

were observed.

In orthopedic trauma surgical treatment, the local application of

phages appears promising. However, the optimal form of application,

required quantities, and the frequency of phage application remain

unclear (4). While some authors advise the use of a draining device

for the administration of phages into the wound cavities, the potential

of the hydrogel as a phage delivery system has been highlighted (6,

18). In vitro and in vivo results of the present case indicate that

application via a hydrogel may bea possible and practical solution.

ese ndings are in line with a previously published case reporting a

salvage therapy in a patient with an infected knee megaprosthesis. e

in vitro testing of the impact of the hydrogel on phage activity showed

a rapid release and also stable titers for at least 6 h indicating

compatibility (8). e present case shows that this innovative approach

is also feasible for the treatment of bone infection and that phages

delivered via hydrogel may oer a promising strategy by targeting

specic bacterial strains. Based on these results, localized and

sustained delivery of phages to the infection site could

bedemonstrated. Furthermore, it can beassumed that the ecacy of

phage therapy may beenhanced by maximizing contact time between

phages and bacteria compared to rinsing the wound cavities with

phages. Another potential implication involves the versatility of

hydrogel delivery systems, which enable the incorporation of other

therapeutic agents, such as antibiotics for a synergistic approach.

Metagenomic sequencing is a powerful tool for identifying and

characterizing bacteriophages. However, its analytical sensitivity poses

challenges in detecting low-abundance bacteriophages directly from

the original clinical sample material (19). Diluting eects are

particularly important for high-volume sample material such as

wound drainage uid or blood. Additionally, the complexity of these

samples, combined with the presence of host DNA, can mask

bacteriophages. erefore, weused specic real-time quantitative PCR

(qPCR) protocols to conrm the presence of phages, ensuring

sensitive detection of bacteriophages. Reduction in bacterial counts

and the stability of phage titers as well as qPCR protocols are

commonly employed to conrm the presence and activity of phages

post-release and from hydrogels (20). ese ndings are in line with

other studies that detected the release of bacteriophages from

hydrogels for several days (21). Nevertheless, detection will depend on

the sensitivity of the detection method and the sample material.

Furthermore, the development and standardization of phage cocktails

pose several challenges. is study highlights the need for rigorous

characterization to ensure the safety and ecacy of these cocktails.

e case presentation also highlights practical weaknesses in the

current therapeutic approach and reveals challenges to beaddressed. (1)

In three performed revision surgeries, dierent communities of

antibiotic-resistant microbes were observed in routine diagnostics

conducted through culture in each operation. is can oen beobserved

in bone and joint infections (22, 23). However, the therapeutic

consequences for antibiotic and alternative therapies, such as phage

application, still need to bedetermined. Very high local antibiotic doses

for MDR bacteria may lead to the initial disappearance of MDR bacteria

and eventual infection eradication (24). (2) Bacteriophage testing on the

identied MDR bacteria did not yield the initially desired results, except

for the Georgian phage cocktail, which demonstrated ecacy against

MDR E. coli. (3) e time elapsed until obtaining results was substantial.

While a 6-week interval between operations is typically required for a

two-stage Masquelet procedure, in this case, the therapeutic interval of

92 days is more than twice as long. erefore, the testing and production

of suitable phages must besignicantly optimized.

6 Conclusion

In conclusion, this case demonstrates the potential of localized

phage therapy delivered via hydrogel in managing complex

fracture-related infections with multi-drug resistant bacteria and

severe bone defects. e integration of phage therapy with

established surgical procedures resulted in successful infection

eradication and bone reconstruction. High-throughput

metagenomic sequencing provided insights into bacteriophage

release dynamics in the wound environment, supporting the

feasibility of sustained delivery for several days aer surgery in a

clinical context. e case study highlights the fact that applying

phages had no negative side eects. It remains uncertain in the

present case whether bacteriophage application has eectively

contributed to preventing reinfection. Despite practical challenges,

including microbial variability and timely production processes,

phage therapy holds promise as a personalized approach for patients

with challenging bone infections.

Data availability statement

e raw data supporting the conclusions of this article will

bemade available by the authors, without undue reservation.

Ethics statement

Ethical approval was not required for the studies involving

humans because this case falls under the category of intended

compassionate use of phages according to the §37 of the Helsinki

Declaration. e studies were conducted in accordance with the local

legislation and institutional requirements. e participants provided

their written informed consent to participate in this study. Written

informed consent was obtained from the participant/patient(s) for the

publication of this case report.

FIGURE4

The release of bacteriophages from hydrogel and the impact of DAC

gel on phages. The scatter dot plot graph was created using

GraphPad Prism 9.5.

Alt et al. 10.3389/fmed.2024.1428432

Frontiers in Medicine 07 frontiersin.org

Author contributions

VA: Conceptualization, Data curation, Formal analysis, Investigation,

Methodology, Validation, Writing – original dra. AG:

Conceptualization, Data curation, Formal analysis, Investigation,

Methodology, Project administration, Validation, Writing – review &

editing. MM: Formal analysis, Investigation, Methodology, Validation,

Writing – review & editing. FH: Investigation, Methodology, Validation,

Writing – review & editing. GM: Data curation, Formal analysis,

Investigation, Methodology, Validation, Writing – review & editing. DP:

Formal analysis, Investigation, Methodology, Validation, Writing –

review & editing. NW: Conceptualization, Investigation, Validation,

Writing – review & editing. J-PP: Investigation, Methodology, Validation,

Writing – review & editing. AH: Formal analysis, Investigation,

Methodology, Validation, Writing – review & editing. MR:

Conceptualization, Data curation, Investigation, Methodology, Project

administration, Validation, Writing – original dra.

Funding

e author(s) declare that no nancial support was received for

the research, authorship, and/or publication of this article.

Acknowledgments

The authors thank Tristan Ferry, Hospices Civils de Lyon,

Lyon, France, for sharing his experiences and providing the

DAC gel.

Conﬂict of interest

e authors declare that the research was conducted in the

absence of any commercial or nancial relationships that could

beconstrued as a potential conict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors

and do not necessarily represent those of their aliated

organizations, or those of the publisher, the editors and the

reviewers. Any product that may beevaluated in this article, or

claim that may bemade by its manufacturer, is not guaranteed or

endorsed by the publisher.

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