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The use of combination therapy for the improvement of colistin activity against bacterial biofilm

November 2023
Brazilian Journal of Microbiology 55(1)

November 2023
55(1)

DOI:10.1007/s42770-023-01189-7

Authors:

Abduladheem Al-Attabi

College of Medicine, University of Thi-Qar, Al-Nasiriyah,Iraq.

Montaha Al-Saffar

Middle Technical University

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Colistin is used as a last resort for the management of infections caused by multi-drug resistant (MDR) bacteria. However, the use of this antibiotic could lead to different side effects, such as nephrotoxicity, in most patients, and the high prevalence of colistin-resistant strains restricts the use of colistin in the clinical setting. Additionally, colistin could induce resistance through the increased formation of biofilm; biofilm-embedded cells are highly resistant to antibiotics, and as with other antibiotics, colistin is impaired by bacteria in the biofilm community. In this regard, the researchers used combination therapy for the enhancement of colistin activity against bacterial biofilm, especially MDR bacteria. Different antibacterial agents, such as antimicrobial peptides, bacteriophages, natural compounds, antibiotics from different families, N-acetylcysteine, and quorum-sensing inhibitors, showed promising results when combined with colistin. Additionally, the use of different drug platforms could also boost the efficacy of this antibiotic against biofilm. The mentioned colistin-based combination therapy not only could suppress the formation of biofilm but also could destroy the established biofilm. These kinds of treatments also avoided the emergence of colistin-resistant subpopulations, reduced the required dosage of colistin for inhibition of biofilm, and finally enhanced the dosage of this antibiotic at the site of infection. However, the exact interaction of colistin with other antibacterial agents has not been elucidated yet; therefore, further studies are required to identify the precise mechanism underlying the efficient removal of biofilms by colistin-based combination therapy.

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Brazilian Journal of Microbiology

https://doi.org/10.1007/s42770-023-01189-7

CLINICAL MICROBIOLOGY - REVIEW

The use ofcombination therapy fortheimprovement ofcolistin

activity againstbacterial bioﬁlm

AbduladheemTurkiJalil1 · RawaaTurkiAbdulghafoorAlrawe2· MontahaA.Al‑Saar3·

MurtadhaLaftaShaghnab4· MunaS.Merza5· MuntherAbosaooda6· RahimLatef7

Received: 14 July 2023 / Accepted: 27 October 2023

Abstract

Colistin is used as a last resort for the management of infections caused by multi-drug resistant (MDR) bacteria. However,

the use of this antibiotic could lead to diﬀerent side eﬀects, such as nephrotoxicity, in most patients, and the high prevalence

of colistin-resistant strains restricts the use of colistin in the clinical setting. Additionally, colistin could induce resistance

through the increased formation of bioﬁlm; bioﬁlm-embedded cells are highly resistant to antibiotics, and as with other anti-

biotics, colistin is impaired by bacteria in the bioﬁlm community. In this regard, the researchers used combination therapy

for the enhancement of colistin activity against bacterial bioﬁlm, especially MDR bacteria. Diﬀerent antibacterial agents,

such as antimicrobial peptides, bacteriophages, natural compounds, antibiotics from diﬀerent families, N-acetylcysteine, and

quorum-sensing inhibitors, showed promising results when combined with colistin. Additionally, the use of diﬀerent drug

platforms could also boost the eﬃcacy of this antibiotic against bioﬁlm. The mentioned colistin-based combination therapy

not only could suppress the formation of bioﬁlm but also could destroy the established bioﬁlm. These kinds of treatments

also avoided the emergence of colistin-resistant subpopulations, reduced the required dosage of colistin for inhibition of

bioﬁlm, and ﬁnally enhanced the dosage of this antibiotic at the site of infection. However, the exact interaction of colistin

with other antibacterial agents has not been elucidated yet; therefore, further studies are required to identify the precise

mechanism underlying the eﬃcient removal of bioﬁlms by colistin-based combination therapy.

Keywords Bioﬁlm· Combination therapy· Colistin

Introduction

Colistin is an antimicrobial agent that is extracted from Pae-

nibacillus polymyxa, which belongs to class E of the poly-

mixin group [1]. Two forms of colistin sulfate and colistin

methane sulfonate are used in oral-topical and injectable

forms for humans, respectively [2]. The general mechanism

and goals of colistin are binding to the negative charge of

lipopolysaccharides (LPS) in the outer membrane of Gram-

negative bacteria, increasing membrane permeability and

bacterial lysis, producing reactive oxygen species, and inhib-

iting respiratory enzymes [3]. In addition to the fact that

some bacteria, such as Proteus species, Morganella mor-

ganii, Serratia species, Providencia species, Burkholderia

pseudomallei, Neisseria species, and Edwardsiella tarda,

are intrinsically resistant to colistin, resistance can be seen

through chromosomal mutations of genes involved in LPS

biosynthesis or glucose transport pathways and the transfer

of the mcr-1 gene through a plasmid [4].

Responsible Editor: Beatriz Ernestina Cabilio Guth

* Abduladheem Turki Jalil

abedalazeem799@gmail.com

1 College ofMedicine, University ofThi-Qar, Al-Nasiriya,

Iraq

2 Education College forWomen, Department ofBiology,

University ofAnbar, Anbar, Iraq

3 Community Health Department, Institute ofMedical

Technology/Baghdad, Middle Technical University,

Baghdad, Iraq

4 National University ofScience andTechnology, Nasiriyah,

DhiQar, Iraq

5 Prosthetic Dental Techniques Department, Al-Mustaqbal

University College, Babylon51001, Iraq

6 Medical Laboratory Technology Department, College

ofMedical Technology, The Islamic University, Najaf, Iraq

7 Medical Technical College, Al-Farahidi University, Baghdad,

Iraq

Brazilian Journal of Microbiology

1 3

Colistin is often used as a last line of defense in critical

clinical conditions such as bacteremia/sepsis and ventilator-

associated pneumonia in the intensive care unit and against

Gram-negative bacteria such as Pseudomonas spp., Acine-

tobacter spp., and Enterobacteriales [5]. According to the

studies, the prevalence of resistance to colistin is increasing,

especially in multi-drugs resistant (MDR) bacteria, and the

scientiﬁc community has demanded to reduce the use of

colistin [1]. In addition, one of the limitations of the wide-

spread use of colistin is the high incidence of poisoning,

such as renal and neurotoxicity, neuromuscular blockade,

and, in some cases, fatal manifestations [6].

The point to consider is that one of the biggest problems

in the treatment of bacterial infections is the formation of

bioﬁlm by the most important pathogens, and the activity of

colistin is often impaired when faced with bioﬁlm [7]. Bio-

ﬁlm formation is a bacterial community in which microor-

ganisms are enclosed in an exopolysaccharide matrix, which

leads to extensive phenotypic changes in the bacterial popu-

lation and makes them 1–1000 times more resistant than

their planktonic form [7, 8].

Noteworthy, a recently published study reported that a

minimum inhibitory concentration (MIC)-dependent con-

centration of colistin could not suppress Escherichia coli

growth. On the other hand, this antibiotic could lead to bac-

terial regrowth, with the possibility of resulting in colistin

resistance. Moreover, increased expression of the phoQ gene

could lead to increased bioﬁlm formation in colistin-induced

resistance bacteria, which prevents colistin from reaching

the site of action, ﬁnally inducing antibiotic resistance [9].

Therefore, in addition to the increased prevalence of colistin-

resistant strains in recent years, the bioﬁlm community could

also decrease the inhibitory activity of this antibiotic.

To this end, researchers have used new therapeutic

options, including combination therapy, to increase the

activity of colistin against bacterial biofilm, especially

MDR isolates [10]. Antimicrobial peptides, bacteriophages,

natural compounds and natural products, antibiotics, N-ace-

tylcysteine, and quorum-sensing inhibitors are among the

agents that have shown promising results when used with

colistin. The purpose of combined treatment is to overcome

resistance to colistin and other antimicrobial agents, increase

the eﬀectiveness of the combination compared to mono-

therapy, and reduce the toxicity associated with colistin [6,

11]. It should be noted that previous studies have shown the

existence of synergistic interactions when combining colistin

with antimicrobial drugs against Gram-negative clinical iso-

lates and reducing hospital mortality [12]. Considering the

therapeutic and practical role of colistin in the clinic and as

the last line of treatment, the purpose of this review study is

to examine and discuss the combined use of this drug along

with other therapeutic agents and drug carriers for inhibition

and destruction of bacterial bioﬁlm in a comprehensive way.

Biolm

Bacterial organisms have been found to undergo evolu-

tionary processes in response to various obstacles and dif-

ﬁculties encountered in hostile settings. These problems

may encompass those that are induced by the existence

of host immunity, the presence of an antimicrobial agent,

and restrictions in nutritional availability. Bioﬁlm pro-

duction is a notable survival strategy employed by bac-

teria [13–15]. A bioﬁlm refers to a complex assemblage

of many microbial organisms that are enveloped inside

a self-generated polymeric matrix. This matrix serves to

anchor the bioﬁlm to either living (biotic) or non-living

(abiotic) surfaces. It is worth noting that the production

of bioﬁlms is a capability exhibited by nearly all bacteria,

given the presence of appropriate environmental condi-

tions [16]. Bacterial aggregation and bioﬁlm maturation

encompass both reversible and irreversible stages, which

are inﬂuenced by numerous conserved and/or species-spe-

ciﬁc variables. During the initial stage, the microorganism

establishes a reversible attachment to a surface by engag-

ing in weak contact, such as van der Waals forces, with

either an abiotic or biotic surface. Various surfaces can

exist in diﬀerent forms, encompassing earth and aquatic

systems as well as indwelling medical equipment. Addi-

tionally, surfaces can be found in living tissues, such as

heart valves, tooth enamel, the lung, and the middle ear

[17]. The second stage involves the process of irreversible

attachment, which is facilitated by ﬂagella, pili, and other

surface appendages, as well as speciﬁc receptors [18].

The accumulation of multilayered cells occurs through

cellular division; after which they initiate the synthesis of

their own extracellular polymeric substance (EPS) matrix.

This matrix mostly consists of polysaccharides, proteins,

and extracellular DNA, which play a crucial role in pro-

moting the formation of the bioﬁlm [19]. The detection of

EPSs can be achieved through microscopic examination as

well as chemical analysis. EPSs serve as the foundational

matrix or structural framework for the formation and main-

tenance of bioﬁlms. EPS has a high level of hydration,

with approximately 98% of its composition consisting of

water. Additionally, EPS displays a strong adherence to the

underlying surface. Furthermore, the EPS bioﬁlm matrix

serves as a protective barrier for microbes, shielding them

from the eﬀects of antimicrobial medications and the host

immune system. Hence, the microorganisms implicated in

co-infections have the ability to form polymicrobial bio-

ﬁlms, which exhibit not only inherent genotypic resistance

but also phenotypic resistance or tolerance to antimicrobial

medicines that are linked to the bioﬁlm matrix [20]. Water

channels are present in a fully developed bioﬁlm, facili-

tating the eﬃcient distribution of nutrients and signaling

Brazilian Journal of Microbiology

1 3

chemicals throughout the bioﬁlm structure. Bioﬁlm cells

exhibit detachment either individually or in aggregates due

to both internal and extrinsic stimuli. Ultimately, these

cells become dispersed, leading to their colonization of

various ecological niches [13].

Bioﬁlm-associated organisms have a reduced growth rate

compared to planktonic organisms, perhaps because of the

constraints imposed by nutrition and/or oxygen deprivation

on the cells [21]. Cell detachment from the bioﬁlm occurs

due to two primary mechanisms: cellular proliferation

and division, or the elimination of bioﬁlm aggregates that

encompass substantial quantities of cells. The potential for

detached cells to induce a systemic infection is contingent

upon various aspects, encompassing the host immune sys-

tem’s reaction [22]. Bioﬁlms hold considerable importance

in the ﬁeld of public health due to the fact that microor-

ganisms associated with bioﬁlms demonstrate a signiﬁcant

reduction in their susceptibility to antimicrobial treatments.

The bioﬁlm represents a suitable niche for the exchange

of resistance genes [17, 23]. The impact on susceptibil-

ity can be categorized as either intrinsic, which refers to

characteristics inherent in the bioﬁlm mode of growth, or

acquired, which is associated with the acquisition of resist-

ance plasmids. There exist a minimum of three rationales

accounting for the inherent antimicrobial resistance exhib-

ited by bioﬁlms. Initially, it is imperative for antimicrobial

drugs to eﬀectively permeate the EPS matrix in order to

establish contact with and neutralize the microorganisms

residing within the bioﬁlm [24]. EPSs inhibit the diﬀusion

process through two mechanisms: chemical reactions with

antimicrobial compounds and restriction of their transport

rate. Additionally, organisms linked with bioﬁlms exhibit

diminished growth rates, resulting in a decreased uptake of

antimicrobial drugs into the cell. Consequently, this phe-

nomenon impacts the kinetics of inactivation [25]. Further-

more, the local environment encompassing the cells inside a

bioﬁlm may oﬀer favorable conditions that serve to enhance

the organism’s protection [25]. In relation to acquired resist-

ance, studies have demonstrated that the exchange of plas-

mids within bioﬁlms can occur under many circumstances.

Numerous bacterial species have demonstrated the ability

to transfer plasmids to diverse bacterial counterparts. The

potential factors contributing to increased plasmid transfer

within bioﬁlms encompass the heightened likelihood of cel-

lular interaction and the little impact of shear pressures on

both the disruption of cell-to-cell contact and the integrity

of the pili essential for conjugation [26].

Finally, it is noteworthy to mention that a crucial aspect of

addressing bioﬁlm is the recognition of polymicrobial bio-

ﬁlm communities that engage in mutual interactions, result-

ing in a synergistic impact. These interactions frequently

result in an elevated level of resistance to both host and

antimicrobial drugs across all participating species [27, 28].

This phenomenon can arise from either heightened tolerance

or adaptive resistance, which stem from the synergistic inter-

actions between diﬀerent species, or from the presence of

antibiotic-resistant organisms inside a polymicrobial bioﬁlm,

which confers protection to other species within the bioﬁlm

against antibiotic interventions. Species inside polymicro-

bial bioﬁlms frequently exhibit heightened pathogenicity, an

enhanced capacity to digest and exploit organic molecules

within their surroundings, and provide a conducive milieu

for the dissemination of adaptive characteristics and genes

associated with antimicrobial resistance, both within and

across species [29, 30]. Due to this feature, the management

of diseases associated with microbial bioﬁlms has emerged

as a highly complex issue within the healthcare system. The

cumulative impact of infections linked to bioﬁlms can be

highly debilitating for patients, as these illnesses have the

ability to persist for extended periods, leading to a loss of

hope for patients regarding their recovery. In particular, bio-

ﬁlm has been identiﬁed in various types of wounds, includ-

ing chronic leg ulcers, diabetic foot ulcers, pressure ulcers,

burns, malignant wounds, and surgical wounds [31–33].

Combination use ofcolistin withother

antibiotics

Researchers are considering combining colistin with other

antibiotics to improve bioﬁlm eradication. Noteworthy, anti-

biotics from diﬀerent families inhibit bacteria using various

mechanisms, and synergy assessment has clariﬁed the inter-

action of two drugs in combination against bacterial isolates.

Therefore, combination therapy could suppress bacterial

communities more eﬃciently in comparison to monotherapy.

To this end, researchers considered colistin-carbapenems

combination therapy to inhibit the bioﬁlm community of

Gram-negative bacteria. In a recently published study, dif-

ferent concentrations of colistin-meropenem showed a syn-

ergistic eﬀect against the bioﬁlm structures of A. baumannii

and P. aeruginosa strains, while an indiﬀerence eﬀect was

found against K. pneumoniae [34]. The results of another

investigation also showed that colistin-meropenem combi-

nation therapy decreases the bioﬁlm formation of Myroides

odoratimimus strains by 92.4%. Noteworthy, colistin showed

a low inhibitory eﬀect against bioﬁlm when used alone,

while the level of inhibition improved approximately three-

fold when used in combination with meropenem or cip-

roﬂoxacin [35]. In line with these ﬁndings, other studies

also reported the synergistic eﬀect of colistin-carbapenems

combination therapy against a bioﬁlm community of diﬀer-

ent bacteria [36–38]. In this regard, Tamayo etal. reported

that the colistin-doripenem combination not only showed

synergistic eﬀects against bioﬁlm-embedded P. aeruginosa

cells but also reduced the emergence of colistin resistance

Brazilian Journal of Microbiology

1 3

strains. The authors proposed that colistin’s ability to destroy

the outer membrane of Gram-negative bacteria enhances

the permeability of these bacteria and could allow greater

access of doripenem to the critical penicillin-binding pro-

teins located on the cytoplasmic membrane, where the car-

bapenems act [38].

On the other hand, the results of a study published in

2019 indicated that the addition of colistin to meropenem

produced no relevant beneﬁts against extended-spectrum-

β-lactamase (ESBL)-producing K. pneumoniae; however,

this combination therapy protected against the emergence

of colistin-resistant subpopulations [39]. Other beta-lac-

tams, such as ceftazidime, also showed a synergistic eﬀect

in combination with colistin against bacterial bioﬁlm. In this

regard, a recently published study reported that colistin mon-

otherapy could signiﬁcantly destroy the bioﬁlm structure of

P. aeruginosa, but monotherapy leads to the emergence of

colistin-resistant strains. Nonetheless, colistin-ceftazidime

combination therapy prevented resistance emergence to

both antibiotics and improved killing in comparison to each

monotherapy [40]. This supports the ﬁnding by Gómez-

Junyent etal., who reported colistin plus meropenem and

ceftolozane/tazobactam as the most applicable therapeutic

approaches for the treatment of MDR and extensively drug-

resistant (XDR) P. aeruginosa bioﬁlm-associated infections,

respectively [41]. Although the interaction of colistin and

beta-lactams against bioﬁlm structure is not well known,

the ﬁndings of previous studies have shown that bacteria

with low metabolic proﬁles within the bioﬁlms are more

susceptible to colistin, while bacteria that are more meta-

bolically active and present in the outer layers of the bioﬁlm

are more susceptible to beta-lactams [40, 42–45]. Therefore,

colistin/beta-lactam combination therapy can eﬃciently kill

both groups of bacteria and reduce the emergence of antibi-

otic resistance. Additionally, as mentioned, colistin destroys

the outer structure of bioﬁlm and facilitates the penetration

of beta-lactams to subpopulations within bioﬁlm layers.

Altogether, colistin and beta-lactam’s functions on various

bioﬁlm layers and cellular pathways could explain the syn-

ergy observed with the combination. Furthermore, colistin/

beta-lactam combination therapy could eﬀectively destroy

the bioﬁlm community of MDR bacteria and reduce the

emergence of colistin-resistant strains. However, it seems

this combination therapy has variable eﬀects against vari-

ous Gram-negative bacteria; thus, further studies are needed

to evaluate whether the beneﬁts of the colistin/beta-lactam

combination therapy are obtained over longer periods or

against diﬀerent carbapenem-resistant or ESBL-producing

bacteria.

In addition to beta-lactams, rifampicin, also in combina-

tion with colistin, showed promising results for the inhibi-

tion of bacterial bioﬁlm [37, 46, 47]. Geladari etal. reported

that colistin plus rifampicin interacted in synergy to decrease

the viability of carbapenem-resistant K. pneumoniae bioﬁlm

cells at low rifampicin concentrations. Notably, the syner-

gies observed with this combination therapy were higher

than those observed with other combinations such as colis-

tin/meropenem and colistin/tigecycline [37]. Of note, the

authors supposed that colistin leads to the disruption of the

outer bacterial membrane and improves the penetration and

intracellular concentration of rifampicin to suppress DNA

transcription or the mutual killing of resistant subpopula-

tions by each drug [37, 48, 49]. Additionally, rifampicin’s

ability to penetrate bioﬁlm cells is supported by clinical

combination studies using rifampicin to treat prosthetic

material infections [49]. The result of another study demon-

strated that polymixin-rifampicin combination therapy, after

one hour, reduced the gene expression of quorum-sensing

(QS)-regulated virulence factors, such as bioﬁlm formation

and secretion systems of P. aeruginosa. On the other hand,

this combination therapy, after four hours, enhanced the

expression of peptidoglycan biosynthesis genes. Notably,

the combination therapy caused a substantial accumulation

of nucleotides and amino acids that lasted at least 4h, indi-

cating that bacterial cells were in a state of metabolic arrest

[46]. Therefore, the colistin-rifampicin combination therapy

had a synergistic eﬀect against the bioﬁlm community of

diﬀerent bacteria at relatively low concentrations, and this

combination therapy should be considered for the manage-

ment of bioﬁlm-associated infection and warrant further

assessment in appropriate invivo models.

In the end, the double combination of colistin and tigecy-

cline also showed promising inhibitory eﬀects against bac-

terial bioﬁlm. Tigecycline, a member of the glycylcycline

class of semisynthetic antimicrobial agents, has the potential

for disruption of bioﬁlm structure; however, the ﬁndings of

the invitro catheter model study showed that tigecycline

monotherapy could be related to the regrowth of bacteria

[50]. To this end, the combination of this antibiotic with

colistin was considered by researchers for better inhibition of

bioﬁlm cells. Sato etal. reported that the bioﬁlm community

of MDR-A. baumannii was eradicated with colistin but not

tigecycline. Noteworthy, the combination usage of colistin

with a high concentration of tigecycline eﬀectively eradi-

cated bioﬁlm, while attenuation happened with the com-

bination of colistin and low concentrations of tigecycline

[51]. In line with these results, another study also reported

that just high concentrations of these drugs could lead to

synergistic eﬀects in the double combination therapy against

K. pneumonia bioﬁlm, whereas at low concentrations, the

combination treatment indicated indiﬀerent results [37]. It

is noteworthy to mention that eﬄux pumps are considered

important factors for virulence and antibiotic resistance

in Gram-negative bacteria [52]. Previous studies reported

that diﬀerent eﬄux pumps such as EmrAB have an impor-

tant role in resistance to colistin, and low concentrations

Brazilian Journal of Microbiology

1 3

of tigecycline lead to the upregulation of this eﬄux pump

[53, 54]. Therefore, the authors suggested that one possible

explanation for this observation might be that a low con-

centration of tigecycline increases the expression of eﬄux

pumps and consequently attenuates the bactericidal activity

of colistin in combination therapy [51].

Collectively, in combination therapy, the second antibi-

otic targeting distinct bacterial subpopulations with diﬀerent

antimicrobial susceptibilities could complete the function

of colistin against bacterial bioﬁlm. In addition to this so-

called subpopulation synergy (where diﬀerent drugs target

cells with diﬀerent susceptibilities), mechanistic synergy

has also been proposed for combinations involving colistin,

whereby each drug acts on diﬀerent metabolic pathways or

otherwise enhances killing by the second drug [38]. How-

ever, the mechanism involved in the synergistic eﬃcacy of

the combination of colistin and other antibiotics is not well

known; therefore, further studies using validated invitro and

animal bioﬁlm models are needed.

Finally, it is worth noting that other antibiotics such as

clarithromycin, levofloxacin, azithromycin, fosfomycin,

minocycline, meﬂoquine, and aminoglycosides were also

used in combination with colistin for the inhibition of bac-

terial bioﬁlms (Table1).

Drug delivery platforms

Colistin, as mentioned, is considered a promising therapeutic

approach for the management of MDR Gram-negative bac-

teria. However, the increased prevalence of colistin-resistant

strains in recent years has restricted the clinical usage of

this antibiotic [84]. Noteworthy, recently published stud-

ies reported that diﬀerent nanoparticles and drug platforms

could be used for diﬀerent clinical purposes [85–87]. To

this end, the use of various drug platforms is suggested by

researchers to deliver intact colistin at the site of infection

and shield its interactions with bacterial bioﬁlm and airway

mucus, thereby enhancing the interaction of this antibiotic

with bacteria.

Sans-Serramitjana etal. proposed that nanoencapsula-

tion could improve the eﬃcacy of colistin against MDR

infections by overcoming the limitations of conventional

pharmaceutical forms. In this concept, these authors evalu-

ated the antibioﬁlm activity of nanostructured lipid carriers

(NLC)—colistin and free colistin against P. aeruginosa. The

ﬁndings indicated the more rapid killing of P. aeruginosa

bacterial bioﬁlms by NLC-colistin than by free colistin [88].

In line with these results, another study that was pub-

lished in 2016 also reported that NLC-colistin had the

same antibacterial function as free colistin against the

planktonic community of P. aeruginosa; nonetheless,

nanoencapsulated colistin was much more eﬃcient in the

eradication of bioﬁlms than free colistin [89]. Further-

more, nano-embedded microparticles (NEMs) for sus-

tained delivery of colistin in the lung were used in another

investigation. To this purpose, the emulsion/solvent diﬀu-

sion technique was used for the production of poly(lactide-

co-glycolide) (PLGA) containing colistin. Noteworthy,

poly (vinyl alcohol) (PVA) and chitosan (CS) were used

to modulate surface properties and enhance the transport

of synthesized nanoparticles through artiﬁcial cystic ﬁbro-

sis (CF) mucus. Additionally, these nanoparticles were

spray-dried in various carriers for the production of MEM.

Colistin-loaded NEM signiﬁcantly removed the bioﬁlm of

P. aeruginosa and showed a prolonged eﬃcacy in bioﬁlm

eradication compared to the free drug. The results of the

confocal analysis conﬁrmed that the antibioﬁlm activity

of nanoparticles could be associated with their ability to

penetrate into bacterial bioﬁlms and to sustain the release

of colistin inside the bioﬁlm community [90].

Notably, the coating of the nanoparticle surfaces with chi-

tosan could facilitate the transport of nanoparticles through

mucus, probably as a consequence of mucus ﬁber collapse,

and produce large channels that can paradoxically enhance

the penetration of cationic chitosan-modiﬁed nanoparticles.

Therefore, the engineering of PLGA nanoparticles contain-

ing colistin should be considered for killing bacterial bio-

ﬁlm, especially in patients with CF [91]. Taken together, the

possible entrapment and slow penetration of colistin within

the bioﬁlm due to electrostatic interactions with the nega-

tively charged alginate matrix can signiﬁcantly decrease the

availability of colistin in the bottom layers of the bioﬁlm. In

this regard, the use of diﬀerent nanoplatforms such as NLC

and PLGA could easily penetrate the bioﬁlm structure; thus,

the colistin-nanoplatform can reach the bacteria located in

the deeper layers of the bioﬁlm faster and more easily than

free colistin [88, 92, 93].

Osteomyelitis, one of the most important local infec-

tions, is mostly caused by Methicillin-resistant Staphylo-

coccus aureus (MRSA); nevertheless, the prevalence of

Gram-negative associated bone infections has signiﬁcantly

increased over the last few years [94]. To this end, diﬀerent

bacteria such as E. coli, P. aeruginosa, and A. baumannii

have attracted much attention owing to their ability to reach

antibiotic multi-resistance. Additionally, bacterial bioﬁlm

leads to antibiotic resistance and is responsible for prolonged

antibiotic treatment and an aggressive surgical approach in

osteomyelitis [95, 96]. As mentioned in the previous parts,

the reports of colistin-resistant bacteria were increased in

recent years, and this drug is not available in bone void ﬁll-

ers for local high-dose delivery [97]. In this regard, the use

of diﬀerent drug platforms was considered by researchers

for the enhancement of colistin’s eﬃcacy against bacterial

bioﬁlm and the improvement of the concentration of this

antibiotic at the site of infection.

Brazilian Journal of Microbiology

1 3

Table 1 Other studies used colistin-based combination therapy for the inhibition of bacterial bioﬁlm

Antibacterial agents Class Bacterium Methods Outcome and comments Reference

OligoG CF-5/20 7Alginate oligomer P. aeruginosa High-molecular-weight alginate polymer

bead and mouse lung infection model

The combination therapy remarkably

decreased the MBEC for colistin.

[55]

Low-molecular-weight alginate oligosac-

charide

Alginate oligosaccharide P. aeruginosa CLSM and SEM evaluation Combining therapy eﬀectively destroyed

both intercolony branching/bridging

and microcolony structures in both non-

mucoid and mucoid models.

[56]

Alginate oligosaccharide Alginate oligosaccharide P. aeruginosa Greiner glass-bottomed optical 96-well

plate, CLSM

This compound signiﬁcantly induced

bacterial death and reduced the formation

of bioﬁlm.

[57]

D-amino acids Amino acid P. aeruginosa CLSM, 96-well plates and bioﬁlm dispersal

assays

The addition of D-amino acids improved

the colistin function and reduced the

count of viable bacteria and MBIC.

[58]

Clarithromycin Antibiotic A. baumannii In vitro antibiotic lock model Colistin-clarithromycin combination

therapy showed bactericidal activity

against bacteria embedded in the bioﬁlm

community.

[59]

Amikacin and levoﬂoxacin Antibiotic P. aeruginosa (CR-isolates) MBECs were examined by counting the

live bacteria in the bioﬁlm, CLSM, and

animal bioﬁlm infection model.

Combined use of colistin with levoﬂoxacin

or amikacin should be considered for

inhibition of bioﬁlm-associated infec-

tions.

[60]

Azithromycin Antibiotic K. pneumonia Bioﬁlm formation in vials and polystyrene

96-well plates

Azithromycin can improve the eﬀectiveness

of colistin.2

[61]

Fosfomycin Antibiotic Diﬀerent GNB Bioﬁlm chequerboard and quantitative

antibioﬁlm assays

Colistin-fosfomycin combination therapy

showed a synergistic eﬀect against the

bioﬁlm community of the majority of

tested strains.

[62]

Fosfomycin Antibiotic CR P. aeruginosa Bioﬁlm formation in polystyrene 96-well

plates

Colistin-fosfomycin combination therapy

showed an inhibitory eﬀect against

bioﬁlm.

[36]

Ceftazidime-avibactam Antibiotic XDR P. aeruginosa MTT Methythiazolyl tetrazolium assay The combination therapy can suppress

the bioﬁlm formation and decrease the

production of drug-resistant bacteria.

[63]

Clarithromycin or esomeprazole Antibiotic K. pneumonia In vitro catheter bioﬁlm model The combination therapy showed a syner-

gistic eﬀect.

[50]

Rifampicin Antibiotic CA A. baumannii Polystyrene microtiter plate bioﬁlm assay Monotherapy was not eﬀective at reducing

bacteria in bioﬁlm, while colistin-

rifampicin combination therapy signiﬁ-

cantly decreased the bacteria.

[47]

Clarithromycin Antibiotic P. aeruginosa In vitro catheter bioﬁlm model The combination therapy was most eﬀec-

tive at reducing bacterial count in bioﬁlm

in comparison to the monotherapy.

[64]

Tobramycin Antibiotic P. aeruginosa Static and dynamic bioﬁlm experiments.

CLSM analysis and lung infection in the

animal model

The antibiotic combination signiﬁcantly

decreased the bacterial count and was

more eﬀective in managing infection in

the animal model than monotherapy.

[65]

Inhaled combination dry powder formula-

tion of colistin and rifapentine

Antibiotic and drug delivery P. aeruginosa Polystyrene microtiter plate bioﬁlm assay Combination dry powder increased the anti-

bacterial against the bioﬁlm community.

[66]

Meﬂoquine Antimalarial medicine P. aeruginosa Bioﬁlm eradication test and bioﬁlm forma-

tion inhibition test

The combination therapy decreased bioﬁlm

formation and removed pre-formed

established bioﬁlms.

[67]

Brazilian Journal of Microbiology

1 3

Table 1 (continued)

Antibacterial agents Class Bacterium Methods Outcome and comments Reference

Nisin Lantibiotics; bacteriocins P. aeruginosa Static microtiter plate assays Nisin could signiﬁcantly reduce the

concentration of colistin for inhibition

of bioﬁlm.

[68]

Enterocin DD14 and nisin Bacteriocins Colistin-resistant E.coli Microtiter plate bioﬁlm assay The combination therapy eradicated bacte-

rial bioﬁlm

[69]

Chitosan-coated human albumin nanopar-

ticles

Drug delivery A. baumannii, K. pneumonia Bioﬁlm formation in polystyrene 96-well

plates

This compound inhibited the formation of

bioﬁlm 4- and 60-fold higher than free

colistin.

[3]

Cephalosporin nitric oxide-donor prodrug Drug delivery P. aeruginosa A crystal violet staining technique and

CLSM

This compound leads to the near-complete

eradication of the bioﬁlm community.

[70]

Polymeric derivative with glyco-polypep-

tide architecture

Drug delivery P. aeruginosa, S. aureus Cr ystal violet method, static chamber

system

This compound leads to the eradication of

established clinically relevant bioﬁlms.

[71]

Clomiphene citrate and Auranoﬁn5FDA-approved drugs P. aeruginosa Formation of bioﬁlms on glass discs and

CLSM

The combination therapy showed antibi-

oﬁlm activity

[72]

Exopolysaccharide biosynthetic glycoside

hydrolases

Glycoside hydrolases P. aeruginosa CLSM, microtiter dish bioﬁlm assay The addition of enzyme to colistin

decreased bacterial count.

[73]

HBED Iron chelators P. aeruginosa Microtiter plate and ﬂow cell bioﬁlm assay Combination therapy signiﬁcantly increased

the eﬀect of colistin microcolony killing

and leads to the almost complete removal

of the bioﬁlm.

[74]

EDTA Iron chelators Colistin-resistant K. pneumonia Crystal violet assay and catheter-related

bioﬁlm infection mouse model

Combination therapy remarkably reversed

colistin resistance in both planktonic and

mature bioﬁlms of colistin-resistant and

eradicated colistin-resistant bacteria from

catheter-related bioﬁlm infections.

[75]

CHIR-090 6LpxC inhibitors P. aeruginosa Bead bioﬁlm assay, bioﬁlms in ﬂow cell

chambers, and mouse bioﬁlm implant

model of infection

The combination therapy, at sub-inhibitory

concentrations, indicated synergistic

activity and suppressed the formation of

bioﬁlm of colistin-tolerant bacteria.

[76]

Maipomycin 1Natural compound A. baumannii MTT assay and CLSM analysis Maipomycin increased the antibioﬁlm

activity of colistin.

[77]

Nutrient dispersion compounds Nutrient dispersion P. aeruginosa CLSM The combination therapy leads to the

remarkably signiﬁcant decrease in the

live bacterial population.

[78]

PFK-158 4PFKFB3 inhibitor Colistin-resistant GNB Bioﬁlm formation assay and SEM The combination therapy signiﬁcantly

reduced bioﬁlm formation and decreased

the cell arrangement density of bioﬁlm.

[79]

Furanone C-30 3QS inhibitors Colistin-resistant GNB Bioﬁlm formation inhibition assays and

standardized invitro bioﬁlm mode

The combination therapy suppressed

the formation of bacterial bioﬁlm and

showed a better eradication eﬀect on

established bioﬁlm than monotherapy.

[80]

N-(2-pyrimidyl) butanamide 8QS inhibitors P. aeruginosa CLSM, 24 well microtiter plates containing

BBM and AHL analogs

Synergistic antibioﬁlm activity was

detected under both anaerobic and aero-

bic conditions.

[81]

Ultrasound patches Ultrasound P. aeruginosa Filter-bioﬁlms Signiﬁcantly enhanced the bacterial killing

of the bioﬁlm community.

[82]

Brazilian Journal of Microbiology

1 3

Aguilera-Correa etal. used bone-targeted mesoporous

silica nanoparticles that were functionalized with gelatin/

colistin coating. The nanoparticles signiﬁcantly reduced

the number of MRSA in the bone just 24h after only one

dose. It is noteworthy to mention that S. aureus by secre-

tion of diﬀerent enzymes, such as cysteine proteases, ser-

ine proteases, and metalloproteases, could degrade gelatin

coating and accelerate the delivery of antibiotics from drug

platforms on the infected bone [98]. In another study, the

authors used mesoporous silica nanoparticles that have been

loaded with moxiﬂoxacin and further functionalized with

colistin and Arabic gum. This nanosystem showed high

aﬃnity toward the bioﬁlm community of E. coli because

of Arabic gum coating and antibacterial activity because of

the colistin disaggregating eﬀect and moxiﬂoxacin bacteri-

cidal eﬀect. The nanosystem, in a short time, could release

large amounts of moxiﬂoxacin; therefore, it could lead to

the preparation of a high concentration of antibiotics nearby

bacteria and in the site of infection from a low quantity of

nanoparticles that might decrease the potential side eﬀects

associated with other administration routes. Additionally,

colistin can directly kill bacteria because this antibiotic is

easily absorbed on the surface of nanoparticles. Interest-

ingly, Arabic gum improves the adsorption capacity of nano-

particles, potentially diminishing the ﬁnal nanoparticle dose

that would be required during the treatment if colistin alone

was to be used [96].

Finally, in a recently published study, microcontainers,

reservoir-based microdevices, were co-loaded with colistin

and ciproﬂoxacin for inhibition of P. aeruginosa bioﬁlm.

The results of this study showed that co-loaded microcon-

tainers are superior to monotherapy and completely killed all

of the bacteria in the planktonic community. Furthermore,

antibiotics in microcontainers work signiﬁcantly faster (just

ﬁve hours) than simple perfusion of antibiotics in bioﬁlm.

The authors proposed that this eﬀect is caused by the burst

release of the antibiotics from the microcontainers, which

creates an immediate high concentration of antibiotics at the

local site of infection and ultimately more destroyed biomass

[99].

Therefore, as mentioned in this section, diﬀerent drug

platforms, such as liposomes, PLGA, and microcontainers,

have an acceptable capacity as delivery systems for inhibi-

tion of bacterial bioﬁlm by reaching immediate high local

colistin concentrations at the site of infection, especially in

patients with CF and osteomyelitis. Additionally, the use

of drug platforms decreases the desired dose of colistin,

therefore reducing the side eﬀects of this antibiotic. Drug

platforms could be functionalized by diﬀerent antibacte-

rial agents such as natural compounds, mucin-degrading

enzymes, metal nanoparticles, and antibiotics; thus, the use

of drug platforms can enhance the antibioﬁlm activity of

colistin. Altogether, drug platforms containing colistin may

Table 1 (continued)

Antibacterial agents Class Bacterium Methods Outcome and comments Reference

Low-frequency ultrasound Ultrasound Pan-resistant bioﬁlms of A. baumannii 24 well microtiter plates Colistin/vancomycin low-frequency ultra-

sound combination therapy decreased the

count of bacteria in bioﬁlms after 8 h and

a continuing decline until 24 h.

[83]

QS, quorum-sensing inhibitor; CR, carbapenem-resistant; MBECs, minimal bioﬁlm eradication concentrations; CLSM, confocal laser-scanning microscopy; DD, disc diﬀusion; GNB, Gram-neg-

ative bacilli; XDR, extensively drug-resistant; MTT, methythiazolyl tetrazolium assay; HBED, N, N'-bis (2-hydroxybenzyl) ethylenediamine-N, N'-diacetic acid (iron chelators); SEM, scanning

electron microscopy; BBM, basic medium for bioﬁlm formation; AHL, N-acyl homoserine lactone

1 This compound was isolated from rare actinomycetes strain Kibdelosporangium phytohabitans XY-R10

2 The authors reported that the DD method and broth growth-based assays may not be good predictors of antibiotic susceptibility in bioﬁlms

3 QS inhibitors

4 6-phosphofructo2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) inhibitor

5 Compounds with antibacterial and antibioﬁlm activities that are commercially available

6 LpxC inhibitors

7 Alginate oligomer

8 QS inhibitors

Brazilian Journal of Microbiology

1 3

be an interesting strategy for the inhibition and removal of

bacterial bioﬁlms; however, further studies are required

to identify the precise mechanism underlying the eﬃcient

removal of bioﬁlms by these platforms.

In the end, other drug platforms, such as colistin-loaded

human albumin nanoparticles, bi-functional alginate oligo-

saccharide–polymixin conjugates, and colistin-conditioned

surfaces, showed promising results for inhibition of bacterial

bioﬁlms (Table1) [3, 57, 100].

N‑Acetylcysteine

N-Acetylcysteine (NAC) is commonly administered as an

antioxidant due to the ability of the free thiol group to react

with nitrogen species and reactive oxygen by constituting

a precursor of intracellular glutathione. Additionally, NAC

is known as a mucolytic agent; therefore, this compound

clears thick mucus from the lungs and is used in combination

therapy with antibiotics for the treatment of lower respira-

tory tract infections [101, 102]. The results of the recently

published studies also showed that NAC could destroy the

matrix architecture of bacterial bioﬁlm and enhance bioﬁlm

breakdown. As mentioned, NAC is a nebulized mucolytic

agent and is used for the management of patients with CF.

These patients are mostly infected with MDR Gram-negative

bacteria such as P. aeruginosa and Stenotrophomonas malt-

ophilia, and the bioﬁlm community of these bacteria is a

main problem in CF patients. Altogether, colistin is used as

a last resort in these patients, and combination therapy with

this antibiotic and NAC for removing MDR bacterial bioﬁlm

could be an applicable therapeutic approach (Fig.1).

In this regard, Aksoy etal. reported that NAC reduced

the minimum bioﬁlm inhibitory concentration (MBIC) of

diﬀerent antibiotics such as colistin against E. coli, Proteus

mirabilis, and Pseudomonas putida [103]. In another experi-

ment, 18 S. maltophilia were collected, and the eﬀect of the

colistin-NAC combination therapy was evaluated against

these bacteria. The combination therapy indicated syner-

gism against the colistin-resistant strains, suggesting that

NAC could antagonize the mechanisms involved in colistin

resistance. Additionally, a dose-dependent potentiation of

colistin activity at sub-MIC concentrations by NAC was also

clearly observed against S. maltophilia bioﬁlms [104]. In

line with these results, Polline and colleagues reported a sig-

niﬁcant synergistic antibioﬁlm function of N-acetylcysteine

(8000mg/L) plus colistin (8mg/L) against colistin-suscep-

tible and colistin-resistant strains [101].

Noteworthy, an artiﬁcial sputum medium (ASM) model

was used in two studies in order to mimic the bacterial bio-

ﬁlm environmental conditions experienced in CF mucus. In

one of these studies, monotherapy with NAC (8000mg/L)

indicated strain-dependent and limited antibioﬁlm activity

against P. aeruginosa. However, the use of colistin (2 to

32mg/L) plus NAC (8000mg/L) demonstrated a relevant

antibioﬁlm synergism against all strains. Furthermore, this

combination therapy also demonstrated a clear synergism

against bacterial bioﬁlms grown in ASM. Noteworthy, the

colistin concentration that allowed observation of synergism

was much higher (i.e., 32 × the MIC) in the ASM model in

comparison to the Nunc-TSP lid system. In this regard, the

authors proposed that the requirement for a higher dosage of

colistin could be due to the strong ionic interactions of this

antibiotic with ASM components such as mucin and extra-

cellular DNA. In addition, the results showed that treatment

of bacterial planktonic cultures with NAC could reduce the

virulence of P. aeruginosa, such as anaerobic respiration,

zinc starvation response, and ﬂagellum-mediated motility.

Hence, pretreatment with NAC could prevent bioﬁlm forma-

tion and lung infection [105].

In line with these results, Aiyer etal. evaluated the

impact of synergistic and additive colistin-NAC combi-

nation therapy in an ASM model using lung macrophages

and bronchial cells to model Achromobacter xylosoxidans

infection. Noteworthy, this bacterium is a Gram-negative

bacillus that has been related to chronic colonization in

CF and antibiotic resistance. The ﬁndings of this study

showed that combination therapy is well tolerated by both

cells and could lead to the synergistic and remarkable

decrease in bacterial counts [106]. These data indicated

that the antibioﬁlm synergism of colistin-NAC combina-

tions against P. aeruginosa strains is also preserved under

environmental conditions mimicking the CF mucus, which

is promising for clinical applications.

Noteworthy, NAC itself can suppress bacterial pro-

liferation and growth by preventing cysteine utilization

by bacterial cells and reducing the formation of EPS and

polysaccharides in many bacterial species [107]. NAC can

destabilize the bioﬁlm structure by interacting with the

main components in the bioﬁlm matrix or by chelating

magnesium and calcium [108]. As mentioned in previous

parts, NAC, at concentrations achievable by topical admin-

istration, could suppress the bioﬁlm community of CF-

associated Gram-negative bacteria and revert the colistin-

resistant phenotype. In this setting, NAC might revert the

colistin resistance phenotype and remarkably improve the

eﬃcacy of this antibiotic against bacterial bioﬁlm. Nev-

ertheless, the report of exact interactions between colistin

and NAC that lead to synergism in combination therapy

is not easy to hypothesize due to the relevant knowledge

gaps on the mechanisms of action of both compounds.

In this regard, further studies with a focus on NAC and

colistin interactions are encouraged because understanding

the mechanisms of such a synergism would be relevant to

the discovery of new antibacterial agents for inhibition of

Brazilian Journal of Microbiology

1 3

MDR bacterial bioﬁlm in diﬀerent infections such as CF

and catheter-associated infections.

Natural compounds

Recently published studies have reported that diﬀerent nat-

ural products or natural compounds (NCs) not only could

reduce the formation of bioﬁlm but also could eradicate

an established bioﬁlm structure [109–111]. Furthermore,

probably a combination treatment of NCs with conven-

tional antibiotics, because of their increasing eﬀective-

ness and potency while minimizing the toxicity and dosage

of antibiotics and reducing the likelihood of developing

resistant strains, should be considered as an applicable

therapeutic approach to manage MDR and bioﬁlm-associ-

ated infection [112]. In this regard, the results of recently

published studies indicated that diﬀerent NCs such as

resveratrol, chrysin, kaempferol, plumbagin, naringenin,

cinnamaldehyde, thymol, and capsaicin could enhance the

eﬃcacy of colistin against bioﬁlms of Gram-negative bac-

teria, even colistin-resistant strains [113–120].

Noteworthy, inhibition of QS in Gram-negative bacte-

ria, especially P. aeruginosa, was considered by research-

ers as a promising therapeutic approach for inhibition

of bacterial bioﬁlm and infections [121]. In this regard,

diﬀerent NCs, such as cinnamaldehyde, could suppress

the expression of Las-, Rhl-, and PQS in P. aeruginosa.

The P. aeruginosa QS system is comprised of three hier-

archically integrated QS systems, Las, Rhl, and PQS, to

manage the expression of diﬀerent virulence factors and

bioﬁlm-related genes of this bacterium [118, 119, 122].

Therefore, NCs, by suppressing the QS system in bacteria,

could inhibit the formation of bacterial bioﬁlm and make

them more susceptible to colistin.

In addition to QS inhibition, NCs could reduce the bio-

ﬁlm formation by inhibition of bacterial attachment in the

ﬁrst step of the switch of planktonic bacteria to a bioﬁlm

phenotype. In this manner, the results of a study showed

that chrysin, a component of honey, could downregulate

the expression of csuA/B and katE [120]. Noteworthy, the

chaperoneusher pili (Csu) assembly system, including

transport proteins (CsuC and CsuD) and pilin subunits

(CsuA/B, CsuA, CsuB, and CsuE), is a main player in A.

baumannii adhesion to the medical appliances surfaces

[120, 123]. Moreover, RpoS has an association with the

formation of established bioﬁlm by inducing motility-

related genes and by suppressing EPS synthesis, and katE

is a reporter gene of RpoS [120, 124, 125]. To this end,

when the expression of the mentioned genes is downregu-

lated by colistin-chrysin combination therapy, the forma-

tion of bioﬁlm is also inhibited.

Furthermore, NCs showed interaction with the outer

membrane and altered the potential of the cell membrane.

Fig. 1 The use of combination therapy for enhancement of colistin activity against bacterial bioﬁlm

Brazilian Journal of Microbiology

1 3

For example, the ﬁndings of a study showed that thymol

can enhance the permeability of membranes to overcome

colistin resistance. Capsaicin can also destroy the perme-

ability of the outer membrane, which could enable hydro-

phobic capsaicin molecules to pass through the LPS in the

outer layer of bacteria and reach their target, thus playing

a synergistic role [113, 114]. Therefore, NCs are permea-

bilizers and should be considered a promising adjuvant to

colistin against Gram-negative bacteria. Finally, it should

be noted that NCs could suppress the expression of eﬄux

pumps, and this possible mechanism should be evaluated in

future studies as a possible synergistic eﬀect in the colistin-

NCs combination therapy [77]. Hence, NCs can improve

the eﬃcacy of colistin against bacterial bioﬁlm and over-

come colistin resistance; therefore, the synergy between

colistin and NCs warrants further experimental evaluation

and conﬁrmation using diﬀerent animal models of infection

and diﬀerent dosage combinations. However, some disad-

vantages, such as poor stability and solubility, hinder the

use of NCs in clinical settings. To this end, the use of drug

delivery platforms can overcome these physicochemical

barriers [119]. Additionally, some of the NCs are used as

antioxidant agents, while the production of reactive oxygen

species (ROS) is one of the important antibacterial mecha-

nisms mediated by colistin [126]. Therefore, co-administra-

tion of NCs that remove ROS could increase the persister

cells, and this issue should be evaluated in further studies.

Antimicrobial peptides

Antimicrobial peptides (AMPs), host defense peptides that

mainly consist of protein fragments, are produced by both

prokaryotes and eukaryotes and act as a ﬁrst line of defense

for the immune system [127]. AMPs are broad-spectrum

antibacterial agents that kill bacteria rapidly with low cyto-

toxicity for eukaryotic cells [127, 128]. Additionally, these

agents have an antibioﬁlm capacity using diﬀerent mecha-

nisms, such as disruption of membrane and EPS, downreg-

ulation of bioﬁlm-associated genes, interference with cell

signaling, and inhibition of stringent response [129].

In this regard, researchers considered colistin-AMP com-

bination therapy a promising antibioﬁlm strategy. Morroni

etal. reported synergic activity for colistin-LL37 combina-

tion therapy against ESBL- and carbapenemase-producing and

mcr-1 carrying E. coli. Noteworthy, AMP LL-37 is a 37-amino

acid peptide that is proteolytically released from the human

cathelicidin hCAP-18. AMP LL-37 signiﬁcantly reduced the

E. coli bioﬁlm at 1 × MIC concentration, and the expression

of mcr1, a colistin-resistant-associated gene, did not aﬀect the

function of this AMP [130]. The results of another study also

showed that a combination of colistin and diﬀerent AMPs

such as LL-37 could decrease minimum bioﬁlm eradication

concentration (MBEC) by eightfold [131]. Other authors also

reported that a combination of the sub-MIC concentration of

CRAMP (a cathelicidin-associated AMP) with colistin indi-

cated interesting synergistic activity against the P. aeruginosa

bioﬁlm community and reduced the expression of QS-regu-

lated genes, resulting in inhibitory eﬀects on QS-regulated

virulence phenotypes (pyocyanin and rhamnolipid) [132].

Furthermore, the ﬁndings of a recently published study dem-

onstrated that colistin, in combination with AMPs, including

citropin 1.1, MP temporin A, and tachyplesin I linear analogue,

have an additive or synergic function in the treatment of pre-

established S. aureus and P. aeruginosa single- and double-

species bioﬁlm [133]. Finally, the combination of colistin with

melittin, an alpha-helical hydrophobic AMP, completely inhib-

ited the bioﬁlm formation of MDR-strong bioﬁlm producer A.

baumannii. Furthermore, the results of this study showed that

melittin decreased the expression of the bap gene. This phe-

nomenon decreases the accumulation of extracellular matrix

in the periphery of bacteria, which consequently enhances the

penetration of antibiotics into the cytoplasm. Besides, it is note-

worthy to mention that melittin can bind to the resistant gene

and inhibit its expression by its DNA binding activity [134].

It seems colistin-AMP combination therapy leads to syn-

ergistic eﬀects due to the diversity of mechanisms of action

found in colistin and AMPs. For instance, colistin, after bind-

ing to the LPS of Gram-negative bacteria, leads to the disrup-

tion of the outer membrane, while AMPs interact with the

cytoplasmic membrane. Therefore, the combination of these

two antibacterial agents causes a perturbation of both the outer

and cytoplasmic membranes and could explain the synergistic

relation between colistin and diﬀerent AMPs such as LL-37

[6, 130, 135]. Additionally, as mentioned, colistin-AMP com-

bination therapy could suppress the bioﬁlm formation in P.

aeruginosa by downregulation of the rhl system gene, result-

ing in decreased expression of pyocyanin and rhamnolipid. It

should be noted that pyocyanin and rhamnolipid are important

virulence factors in P. aeruginosa that are mainly regulated by

the rhl system and manage the secretion of the extracellular

DNA in bacteria (eDNA) and play a vital role in the formation

and diﬀusion of bioﬁlm [132, 136, 137]. Therefore, AMPs

could destroy the structure of bioﬁlm by downregulating the

QS-associated gene and consequently improve the interactions

of colistin with bioﬁlm. However, the studies in this ﬁeld are

very limited, and further invivo studies are needed to evaluate

the synergistic activity of colistin and AMPs and determine the

possible clinical dose through pharmacokinetic experiments.

Phage

Bacteriophages (phages), viruses that infect bacteria, are

considered by scientists as an applicable approach for the

management of MDR bacteria. Additionally, recent studies

Brazilian Journal of Microbiology

1 3

have reported phages as antibacterial agents with inhibitory

eﬀects against bacterial bioﬁlm. However, some disadvan-

tages, such as the narrow host range and the development

of phage resistance, restrict the clinical usage of phages

[138]. To this end, the combination of phages and antibiot-

ics was considered by scientists to inhibit bacterial infection.

Colistin is one of these antibiotics that showed promising

inhibitory eﬀects against MDR bacterial bioﬁlm when used

in combination with phages and phage-derived enzymes.

Vashisth etal. evaluated the synergistic eﬀects of Myophage

φAB182 in combination with diﬀerent antibiotics such as

colistin against the bioﬁlm community of MDR-A. bauman-

nii. The results of this study showed that φAB182 has the

highest synergy with colistin in comparison to other anti-

biotics such as ceftazidime, polymixin B, and cefotaxime.

Furthermore, colistin-phage combination therapy also eradi-

cated the bioﬁlm of A. baumannii [139].

In line with these results, another investigation also

reported that colistin-phage T1245 combination therapy

not only could reduce bacterial density up to approximately

80% but also could reduce biomass and bacterial viability in

3-day established bioﬁlms. After the combination therapy,

scanning microscopic evaluation showed visible alterations

in cell morphology, with membrane poration and cell lysis as

indicated by the presence of cell debris [140]. vWU2001 was

another phage that, in combination with colistin, remarkably

inhibited carbapenem-resistant A. baumannii in comparison

to monotherapy. This combination therapy also leads to a

signiﬁcantly greater enhancement in G. mellonella survival

and in bacterial clearance, as compared with that of phage or

colistin alone [141]. Therefore, the use of colistin in combi-

nation with phages could be used for the inhibition of MDR-

A. baumannii bioﬁlm. The exact interaction of phages and

colistin is not yet elucidated, but some possible mechanisms

have been reported by recent studies. For instance, colistin

interaction with the outer membrane of Gram-negative bac-

teria could increase both phage adsorption and phage DNA

injection [142]. Phage-antibiotic combination therapy may

lead to changes in bacterial morphology, rapid cell lysis, and

phage maturation [143]. Additionally, colistin could cause

cell clustering, and this phenomenon enhanced the phage’s

ability to travel on the adjoined cell surface, increasing

phage infection eﬃciency [144].

It is noteworthy to mention that phages produce the

endolysins during the ﬁnal stages of the replication cycle to

cleave the bacterial cell wall and produce progeny virions.

Endolysins showed good inhibitory functions against Gram-

positive bacteria; however, the presence of the outer mem-

brane restricted their function against Gram-negative bacte-

ria [145, 146]. In this regard, colistin could be employed to

assist the endolysins in overcoming the impenetrability of

the outer membrane [146]. Finally, depolymerases that are

encoded by phages are responsible for destroying EPS, LPS,

and capsular polysaccharides (CPS) of the host bacteria dur-

ing phage invasion. Therefore, phages can destroy bioﬁlm

structure and improve the penetration of the antibiotic to

the deeper layers of bioﬁlm by inducing the synthesis of

enzymes such as polysaccharide depolymerase [147]. Hen

etal. used depolymerase Dpo71, derived from a baumannii

phage, in combination with colistin, for inhibition of the

bioﬁlm community of MDR-A. baumannii. Dpo71 improved

the inhibitory eﬀect of host immune cell activity against

bacteria and also acts as an adjuvant to assist or improve

the function of colistin. An exact evaluation indicated that

the enhanced bactericidal eﬀect of colistin is attributed to

the improved outer membrane destabilization capacity and

binding rate to bacteria after stripping oﬀ the bacterial cap-

sule by Dpo71. Moreover, the combination of Dpo71 could

remarkably improve the colistin activity against bioﬁlm and

enhance the survival rate of A. baumannii-infected Galleria

mellonella. The results of the microscopy evaluation showed

that Dpo71 could signiﬁcantly destroy the bioﬁlms but was

not able to decrease the count of viable cells in the bioﬁlms.

Colistin-Dpo71 combination therapy signiﬁcantly reduced

the count of viable bacterial cells and residual biomass of

bioﬁlm compared with monotherapy. It seems that the antib-

ioﬁlm activity of his combination therapy is associated with

the improved colistin penetration within the bioﬁlm matrix

after the EPS depolymerization by the Dpo71 [148].

Therefore, the combined use of colistin and phage depoly-

merase should be considered for the inhibition of the bioﬁlm

of MDR bacteria. However, knowledge of the exact interac-

tion of extracellular structures of Gram-negative bacteria

cleavage by phage depolymerases is still largely missing.

Additionally, the bioﬁlm’s susceptibility to phage depoly-

merase treatments varied, depending on the bacterial strains,

the activity of the depolymerase enzyme, and the type of

bacteria. Therefore, although colistin-phage combination

therapy may represent promising treatment strategies for

managing MDR bacteria, more conﬁrmatory studies in this

ﬁeld are required.

Conclusion

As mentioned in the previous sections, the use of combina-

tion therapy could boost the inhibitory eﬀects of colistin

against the MDR bacterial bioﬁlm community. To this end,

the use of diﬀerent antibacterial agents, antibiotics, and drug

platforms in combination with colistin could suppress bio-

ﬁlm formation and remove established bioﬁlm. Additionally,

combination therapy reduces the chance of acquiring colistin

resistance and decreases the required dose of this antibiotic

at the site of infection. Therefore, colistin-based combination

therapy could open new frontiers in the treatment of bioﬁlm-

related infections; however, the exact mechanism underlying

Brazilian Journal of Microbiology

1 3

the eﬃcient removal of bioﬁlms by combination therapy has

not yet been reported. To this end, further investigation of

preclinical and clinical studies as well as bio-toxicity evalu-

ation for the human body is needed before the clinical usage

of colistin-based combination therapy for the management

of bioﬁlm-associated infections.

Abbreviations MDR:Multi-drug resistant; LPS:Lipopolysaccharides;

MIC:Minimum inhibitory concentration; ESBL:Extended-spectrum-

β-lactamase; XDR:Extensively drug-resistant; QS:Quorum sensing;

MRSA:Methicillin-resistant Staphylococcus aureus; NAC:N-acetyl-

cysteine; CF:Cystic ﬁbrosis; MBIC:Minimum bioﬁlm inhibitory con-

centration; ASM:Artiﬁcial sputum medium; NCs:Natural compounds;

MBEC:Minimum bioﬁlm eradication concentration

Acknowledgements We greatly appreciate the input from the BioRender

team (BioRender.com) for their collaboration with us in the ﬁgure design.

Author contribution AJ conceived and designed the study. RA, MA

MS, MM, MA, and RL contributed to comprehensive research and

wrote the paper. Notably, all authors have read and approved the

manuscript.

Data availability The authors conﬁrm that the data supporting the ﬁnd-

ings of this study is available within the article.

Declarations

Ethics approval and consent to participate Not applicable.

Consent for publication Not applicable.

Conflict of interest The authors declare no competing interests.

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The Potentiation Activity of Azithromycin in Combination with Colistin or Levofloxacin Against Pseudomonas aeruginosa Biofilm Infection

Article

Full-text available

Mar 2024

Objective Pseudomonas aeruginosa (PA) often displays drug resistance and biofilm-mediated adaptability. Here, we aimed to evaluate the antibiofilm efficacy of azithromycin-based combination regimens. Methods Minimum inhibitory concentrations (MICs), minimal biofilm eradication concentrations (MBECs), and MBEC-combination of azithromycin, colistin, amikacin, and levofloxacin to bioluminescent strain PAO1 and carbapenem-resistant PAO1 (CRPAO1) were assessed. An animal biofilm infection model was established and detected using a live animal bio-photonic imaging system. Results In vitro, PAO1 and CRPAO1 were susceptible to colistin, amikacin, and levofloxacin, while they were unsusceptible to azithromycin. The combinations based on azithromycin have no synergistic effect on biofilm in vitro. In vivo, azithromycin plus colistin or levofloxacin could shorten the PAO1 biofilm eradication time, which totally eradicates the biofilm in all mice on the 8th or 6th day, while monotherapy only eradicate biofilm in 70% or 80% mice on the 8th day. For CRPAO1 biofilm, only azithromycin–colistin combination and colistin monotherapy eradicated the bacteria in 60% and 40% of mice at the 6th day. Conclusion Azithromycin-based combinations containing levofloxacin or colistin had no synergistic effect in vitro, and they are promising for clinical applications due to the good synergistic activity against PAO1 biofilms in vivo.

ABSTRACT IDENTIFICATION AND CHARACTERIZATION OF MSAB GENE INVOLVED IN BIOFILM FORMATION AND VIRULENCE IN STAPHYLOCOCCUS AUREUS

Thesis

Full-text available

Aug 2014

Amelsaad B. Elbarasi

Staphylococcus aureus is an important human pathogen that causes a wide variety of life-threatening infections ranging from minor skin and oral infections to severe infections, such as bacteremia, pneumonia, osteomyelitis, or endocarditis due to the presence and secretion of a large number of virulence factors that are controlled by global virulence regulators in complex networks. Furthermore, S. aureus infections have become a threat to public health because of their high potential to form biofilm, and their ability to resist a wide range of antibiotics has exacerbated further. Therefore, understanding the regulatory networks and developing a drug targeting these networks has the potential to stand as therapeutic targets for future treatment of antibiotic resistant infections. In a previous study msaC was identified as the modulator of sarA, a new global virulence regulator that controls the expression of sarA and biofilm development. Furthermore, it has also been shown that msaC is a part of four-gene operon, msaABCR operon, which includes four-genes: SAUSA300_1296 (msaA), SAUSA300_1295 (msaB), SAUSA300_1294 (msaC), and antisense RNA, msaR. The mechanism of regulation of msaABCR operon and the function of individual genes were not clearly known yet. This study defines the role of msaB, the second gene of the msaABCR operon, which will help shed some light on the regulation of msaABCR. We deleted msaB gene from USA300_LAC, and studied the major msaB phenotypes: pigmentation, protease production, biofilm formation, and rate of cell death. Deletion of msaB

Water table level controls methanogenic and methanotrophic communities and methane emissions in a Sphagnum -dominated peatland

Article

Full-text available

Sep 2023

Peatlands are important sources of the greenhouse gas methane emissions equipoised by methanogens and methanotrophs. However, knowledge about how microbial functional groups associated with methane production and oxidation respond to water table fluctuations has been limited to date. Here, methane-related microbial communities and the potentials of methane production and oxidation were determined along sectioned peat layers in a subalpine peatland across four Sphagnum -dominated sites with different water table levels. Methane fluxes were also monitored at these sites. The results showed that mcrA gene copies for methanogens were the highest in the 10- to 15-cm peat layer, which was also characterized by the maximum potential methane production (24.53 ± 1.83 nmol/g/h). Copy numbers of the pmoA gene for type Ia and Ib methanotrophs were enriched in the 0–5 cm peat layer with the highest potential methane oxidation (43.09 ± 3.44 nmol/g/h). For the type II methanotrophs, the pmoA gene copies were higher in the 10- to 15-cm peat layer. Hydrogenotrophic methanogens and type II methanotrophs dominated the methane functional groups. Deterministic process contributed more to methanogenic and methanotrophic community assemblages in comparison with stochastic process. The level of water table significantly shaped methanogenic and methanotrophic community structures and regulated methane fluxes. Compared with vascular plants, Sphagnum mosses significantly reduced the methane emissions in peatlands. Collectively, these findings enhance a comprehensive understanding of the effect of the water table level on methane functional groups, with consequential implications for reducing methane emissions within peatland ecosystems. IMPORTANCE The water table level is recognized as a critical factor in regulating methane emissions, which are largely dependent on the balance of methanogens and methanotrophs. Previous studies on peat methane emissions have been mostly focused on spatial-temporal variations and the relationship with meteorological conditions. However, the role of the water table level in methane emissions remains unknown. In this work, four representative microhabitats along a water table gradient in a Sphagnum -dominated peatland were sampled to gain an insight into methane functional communities and methane emissions as affected by the water table level. The changes in methane-related microbial community structure and assembly were used to characterize the response to the water table level. This study improves the understanding of the changes in methane-related microbial communities and methane emissions with water table levels in peatlands.

Colistin Resistance and Management of Drug Resistant Infections

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Full-text available

Dec 2022
CAN J INFECT DIS MED

Colistin resistance is a globalized sensible issue because it has been considered a drug of the last-line resort to treat drug-resistant bacterial infections. The product of the mobilized colistin resistance (mcr) gene and its variants are the significant causes of colistin resistance, which is emerging due to the frequent colistin use in veterinary, and these genes circulate among the bacterial community. Apart from mcr genes, some other intrinsic genes and proteins are also involved in colistin resistance. Researchers focus on the most advanced genomics (whole genome sequencing), proteomics, and bioinformatics approaches to explore the question of colistin resistance. To combat colistin resistance, researchers developed various strategies such as the development of newer drugs, the repurposing of existing drugs, combinatorial treatment by colistin with other drugs, a nano-based approach, photodynamic therapy, a CRISPRi-based strategy, and a phage-based strategy. In this timeline review, we have discussed the development of colistin resistance and its management in developing countries.

Resveratrol Increases Sensitivity of Clinical Colistin-Resistant Pseudomonas aeruginosa to Colistin In Vitro and In Vivo

Article

Full-text available

Dec 2022

Infections caused by colistin-resistant P. aeruginosa strains pose a serious threat to public health. It is therefore urgent to find new strategies to deal with these bacterial infections. We aimed to investigate the efficacy and mechanisms of the colistin/resveratrol combination in eradicating colistin-resistant P. aeruginosa isolates and their biofilms both in vitro and in vivo. The results revealed that six clinically isolated colistin-resistant P. aeruginosa strains were multidrug resistant (MDR) strains, and resveratrol showed no antimicrobial activity against eight P. aeruginosa strains. Checkerboard assay and time-kill assays indicated that the combination therapy of resveratrol and colistin indicated a remarkable synergistic effect in vitro, and biofilm assays and SEM indicated synergistic antibiofilm activity. Furthermore, this combination could efficiently eliminate MDR bacteria in a murine infection model and improve the survival rate of Galleria mellonella. Fluorescence analysis, ALP, and β-galactosidase activity test results indicated that the colistin/resveratrol combination increased the membrane permeability of bacteria. In conclusion, our results may provide an efficient alternative pathway against colistin-resistant P. aeruginosa infections. IMPORTANCE P. aeruginosa is a ubiquitous Gram-negative opportunistic pathogen associated with a wide array of life-threatening acute and chronic infections. However, the improper and excessive use of antibiotics has contributed to the increasing emergence of multidrug-resistant (MDR) P. aeruginosa, even colistin-resistant strains, which presents a major challenge to clinical anti-infection treatment. Resveratrol, a naturally occurring polyphenolic antioxidant, can effectively slow down or avoid the occurrence and development of bacterial resistance and is expected to offer a promising strategy to overcome bacterial infections. In this study, colistin/resveratrol combination could synergistically damage the bacterial cell membrane, thereby inducing cell lysis while addressing the emergence of drug resistance. Moreover, this combination therapy may provide an efficient alternative pathway to combat the colistin-resistant P. aeruginosa in clinical practice.

Immunoinformatics design of multi-epitope peptide-based vaccine against Haemophilus influenzae strain using cell division protein

Article

Full-text available

Nov 2022

Haemophilus influenzae is a pathogen that causes invasive bacterial infections in humans. The highest prevalence lies in both young children and adults. Generally, there are no vaccines available that target all the strains of Haemophilus influenzae. Hence, the purpose of this research is to employ bioinformatics and immunoinformatics approaches to design a Multi-Epitope Vaccine candidate employing the pathogenic cell division protein FtsN that specifically combat all the Haemophilus influenzae strains. The current research focuses on developing subunit vaccine in contrast to vaccines generated from the entire pathogen. This will be accomplished by combining multiple bioinformatics and immunoinformatics approaches. As a result, prospective T cells (helper T lymphocyte and cytotoxic T lymphocytes) and B cells epitopes were investigated. The human leukocyte antigen allele having strong associations with the antigenic and overlapping epitopes were chosen, with 70% of the total coverage of the world population. To construct a linked vaccine design, multiple linkers were used. To increase the immunogenic profile, an adjuvant was linked using EAAAK linker. The final vaccine construct with 149 amino acids was obtained after adjuvants and linkers were added. The developed Multi-Epitope Vaccine has a high antigenicity as well as viable physiochemical features. The 3D conformation was modeled and undergoes refinement and validation using bioinformatics methods. Furthermore, protein–protein molecular docking analysis was performed to predict the effective binding poses of Multi-Epitope Vaccine with the Toll-like receptor 4 protein. Besides, vaccine underwent the codon translational optimization and computational cloning to verify the reliability and proper Multi-Epitope Vaccine expression. In addition, it is necessary to conduct experiments and research in the laboratory to demonstrate that the vaccine that has been developed is immunogenic and protective.

The Efficacy of an N-Acetylcysteine–Antibiotic Combination Therapy on Achromobacter xylosoxidans in a Cystic Fibrosis Sputum/Lung Cell Model

Article

Full-text available

Nov 2022

Cystic fibrosis (CF) is a disorder causing dysfunctional ion transport resulting in the accumulation of viscous mucus. This environment fosters a chronic bacterial biofilm-associated infection in the airways. Achromobacter xylosoxidans, a gram-negative aerobic bacillus, has been increasingly associated with antibiotic resistance and chronic colonisation in CF. In this study, we aimed to create a reproducible model of CF infection using an artificial sputum medium (ASMDM-1) with bronchial (BEAS-2B) and macrophage (THP-1) cells to test A. xylosoxidans infection and treatment toxicity. This study was conducted in three distinct stages. First, the tolerance of BEAS-2B cell lines and two A. xylosoxidans strains against ASMDM-1 was optimised. Secondly, the cytotoxicity of combined therapy (CT) comprising N-acetylcysteine (NAC) and the antibiotics colistin or ciprofloxacin was tested on cells alone in the sputum model in both BEAS-2B and THP-1 cells. Third, the efficacy of CT was assessed in the context of a bacterial infection within the live cell/sputum model. We found that a model using 20% ASMDM-1 in both cell populations tolerated a colistin–NAC-based CT and could significantly reduce bacterial loads in vitro (~2 log10 CFU/mL compared to untreated controls). This pilot study provides the foundation to study other bacterial opportunists that infect the CF lung to observe infection and CT kinetics. This model also acts as a springboard for more complex co-culture models.

The Antibacterial Activity of Kaempferol Combined with Colistin against Colistin-Resistant Gram-Negative Bacteria

Article

Full-text available

Oct 2022

Social hygiene is seriously threatened by the rise in colistin (COL) resistance against Gram-negative bacteria (GNB). With resistance to last-line antibiotics such as COL becoming more common, it is imperative to identify alternative treatment options. In our work, we sought to determine if COL plus kaempferol (KP) present synergistic effects on the antibacterial and antibiofilm activities against colistin-resistant (Col-R) GNB in vivo and in vitro. Twenty-four Col-R GNB were collected as the experimental strains. The synergistic activity of COL and KP was evaluated by checkerboard method, time-killing assays, and the Galleria mellonella experiment. The antibiofilm effectiveness of the COL/KP combination against Col-R GNB was assessed using biofilm inhibition and eradication assays and scanning electron microscopy (SEM). Cytotoxicity tests were performed to detect the toxicity of KP monotherapy or combination therapy. There is synergistic antibacterial activity of COL and KP combination in vitro. KP combined with COL could inhibit the formation of bacterial biofilms. The amalgamation of COL and KP considerably reduced the amount of bacteria in the biofilm, according to the SEM findings. The COL/KP combination improved the survivorship of infected larvae in the G. mellonella in vivo infection model. In addition, the combination of KP and COL showed no cytotoxicity at synergistic combined concentrations according to cytotoxicity assays. This represents the first account of the antibacterial and antibiofilm activities of KP in combination with COL against Col-R GNB. Therefore, our results may provide an effective alternative route to combat Col-R GNB infections. IMPORTANCE COL is one of the few antibiotics effective against clinical isolates of GNB. However, in recent years, GNB resistance to colistin has been increasing. As a result, the combined application of colistin in conjunction with nonantibacterial medications has garnered considerable interest. In this work, the KP/COL combination showed effective antibacterial and antibiofilm activities in vitro and in vivo. The synergistic effect of combined application may be attributed to membrane permeability. Due to the low cytotoxicity of the combined concentration, the combination exhibits a promising future for use in clinical anti-infection treatments. This finding might broaden the potential applications for COL.

Electrospun Fibers with Lactobacillus Acidophilus: A Poten- tial In Vitro Solution Against Gardnerella Infections

Article

Jun 2023

Interaction of lytic phage T1245 with antibiotics for enhancement of antibacterial and anti-biofilm efficacy against multidrug-resistant Acinetobacter baumannii

Article

Jan 2023
BIOFOULING

Biofilms associated with multidrug-resistant (MDR) Acinetobacter baumannii on medical devices remain a big clinical problem. Antibiotic susceptibility tests were performed with eight commonly employed antibiotics against clinical isolates. The effects of antibiotics in combination with well-characterized lytic phage T1245 were studied to assess their antibacterial and anti-biofilm efficacy. Ceftazidime, colistin, imipenem, and meropenem significantly reduced bacterial density up to approximately 80% when combined with phage T1245, compared with control. Phage T1245 in combination with ceftazidime, colistin, and meropenem at subinhibitory concentrations demonstrated significant reduction in biomass and bacterial viability of 3-day established biofilms, compared with antibiotic alone. In addition, electron microscopy further confirmed the disruption of biofilm structure and cell morphology upon treatment with phage T1245 and antibiotics, including ceftazidime, colistin, and meropenem. Combined treatment of phage T1245 with these antibiotics could be employed for the management of A. baumannii infections and eradication of the bacterial biofilms.

Antibiotic delivery from bone-targeted mesoporous silica nanoparticles for the treatment of osteomyelitis caused by methicillin-resistant Staphylococcus aureus

Article

Oct 2022
ACTA BIOMATER

Osteomyelitis is a hard-to-treat infection of the bone and bone marrow that is mainly caused by Staphylococcus aureus, with an increasing incidence of methicillin-resistant S. aureus (MRSA). Owing to the aggressiveness of these bacteria in colonizing and destroying the bone, systemic antibiotic treatments fail to eradicate the infection. Instead, it normally entails surgery to remove the dead or infected bone. In this work, we report bone-targeted mesoporous silica nanoparticles for the treatment of osteomyelitis. The nanoparticles have been engineered with a functional gelatine/colistin coating able to hamper premature release from the mesopores while effectively disaggregating the bacterial biofilm. Because antibiotic resistance is a global emergency, we have designed two sets of identical nanoparticles, carrying each of them a clinically relevant antibiotic, that have demonstrated to have synergistic effect. The bone-targeted nanoparticles have been thoroughly evaluated in vitro and in vivo, obtaining a notable reduction of the amount of bacteria in the bone in just 24 h after only one dose, and paving the way for localized, nanoparticle-mediated treatment of MRSA-caused osteomyelitis. Statement of significance In this work, we propose the use of bone-targeted mesoporous silica nanoparticles to address S. aureus-caused osteomyelitis that render synergistic therapeutic effect via multidrug delivery. Because the bacterial biofilm is responsible for an aggressive surgical approach and prolonged antibiotic treatment, the nanoparticles have been functionalized with a functional coating able to both disaggregate the biofilm, hamper premature antibiotic release and protect the intact bone. These engineered nanoparticles are able to effectively target bone tissue both in vitro and in vivo, showing high biocompatibility and elevated antibacterial effect.

The use of combination therapy for the improvement of colistin activity against bacterial biofilm

Abstract

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