Bacteriophage biosensors for antibiotic-resistant bacteria

Bacteriophage biosensors forantibiotic-resistant bacteriaExpert Rev. Med. Devices 11(2), 175–186 (2014)

Irina Sorokulova1,Eric Olsen2 andVitaly Vodyanoy*1

1Department of Anatomy, Physiology,

and Pharmacology, Auburn AL 36849,

USA2US Air Force School of Aerospace

Medicine Epidemiology Laboratory

Wright-Patterson Air Force Base, OH

45433, USA

*Author for correspondence:

Tel.: +1 334 844 5405

Fax: +1 334 844 5388

[email protected]

An increasing number of disease-causing bacteria are resistant to one or more anti-bacterialdrugs utilized for therapy. Early and speedy detection of these pathogens is therefore veryimportant. Traditional pathogen detection techniques, that include microbiological andbiochemical assays are long and labor-intensive, while antibody or DNA-based methodsrequire substantial sample preparation and purification. Biosensors based on bacteriophageshave demonstrated remarkable potential to surmount these restrictions and to offer rapid,efficient and sensitive detection technique for antibiotic-resistant bacteria.

KEYWORDS: detection • filamentous • lytic • monolayers • reporter phages • spheroids

Emergence of antibiotic-resistant bacteria isone of the most serious threats for healthcareand public safety worldwide. CDC estimatesthat more than two million people in the USAare sickened every year with antibiotic-resistantinfections and 23,000 dying as a result ofthese infections [1]. The total economic cost ofantibiotic resistance to US economy is esti-mated near $35 billion a year. The develop-ment of resistance to clinically importantantibiotics in potential biowarfare bacteria hasbecome a serious problem. Conventionalmethods for pathogen detection are time-con-suming, require high-skilled personnel andhave limited application. There is an urgentneed in new real-time detection systems forreliable recognition of antibiotic-resistantpathogens. Bacteriophages (viruses of bacteria)are unique class of naturally evolved recogni-tion probes for bacteria. Bacteriophages areubiquitous in all natural environments, and itis possible to isolate bacteriophage against anyof target bacteria. They are highly specific tobacterial host and stable in wide range of envi-ronmental conditions. So, bacteriophages canbe effectively used in various biosensors as rec-ognition elements for detection of antibiotic-resistant pathogens.

A recent list of antibiotic-resistant bacteriareleased by the CDC includes the followingmicroorganisms: Acinetobacter, Bacillus anthra-cis, Neisseria gonorrhoeae, Group B Streptococcus,Klebsiella, Methicillin-resistant Staphylococcusaureus (MRSA), Neisseria meningitidis, Strepto-coccus pneumoniae, Mycobacterium tuberculosis,

Salmonella Typhi, vancomycin-resistant Entero-cocci (VRE) and vancomycin-intermediate/resistant S. aureus. Pathogen detection bybacteriophage-based sensors including detec-tion platforms, phage immobilization techni-ques and phage engineering were recentlyreviewed [2,3]. This review will be focused pri-marily on the bacteriophages that recognizeantibiotic-resistant bacteria and phage-baseddetection of these pathogens.

BacteriophagesBacteriophages (phages) are viruses of bacteria.Like other viruses, they are absolute parasites.But, they exclusively infect bacteria and do notaffect human or animal cells. Bacteriophagesare ubiquitous in all natural environments andrepresent the most abundant living entities onthe Earth with an estimated 1032 bacterio-phages in total [4]. Phages are extremely het-erogeneous in their structural, physicochemicaland biological properties. More than 95% ofknown phages are tailed bacteriophages, therest are polyhedral, filamentous and pleomor-phic with double-stranded DNA, single-stranded DNA, double-stranded RNA orsingle-stranded RNA genome. Bacteriophagescan be additionally classified according to thestrategy of escaping their hosts: lytic phagesand filamentous phages. Lytic phages producelytic enzymes to destroy the bacterial cell wall,resulting in bacteriolysis. Filamentous phagescan extrude from bacterial cell without fatallysis of the host. Bacteriophages are highlyspecific and can recognize only bacteria of

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one species or only one particular strain. Specificity of bacterio-phage recognition is due to its binding to a specific receptorlocated on the host surface. After the phage recognizes this spe-cific receptor, it binds to the bacterial cell and infects it.

Detection of antibiotic-resistant bacteria with bacterio-phage biosensorsA biosensor is composed of a physical transducer (electrochemi-cal, optical, mass and thermal) and a certain bioprobe (anti-body, aptamers, enzymes, nucleic acids, receptors and wholecells) where biointeraction between the probe and target ofinterest is converted to a significant signal by the physicaltransducer [5–8]. It is obvious that the rapid and correct recogni-tion of bacterial pathogens are essential to be able to preventthe spread of infections and reduce their implications byemploying relevant control techniques. This is an especiallysignificant issue for hospitals, care centers and in the foodindustry. A number of bacteriophages are already used formodern technology advancement to allow the rapid recognitionof pathogen in complex conditions, particularly by usingbacteriophage-based probes [2].

Phages as a recognition probeMany bioassays and biosensors rely on very specialized, sensitiveand selective antibodies as recognition elements [9]. Even thoughantibodies often possess the needed sensitivity and selectivity,their application is restricted by many factors. By way of exam-ple, the binding properties of antibodies can be limited as aresult of adverse environmental conditions [10]. Furthermore,production of polyclonal antibodies needs a process that ishighly time and labor-intensive and can result in a variableproduct. Manufacturing of monoclonal antibodies is normallyeven more complicated and costly. These limitations can beresolved by utilizing bacteriophage or their coat proteins as rec-ognition elements for biosensors [3,11,12]. Both lytic and filamen-tous phages offer extensive libraries to recognize proteinsinteracting with molecular targets. In phage display, the phagefilament is the scaffold for random peptides that are joined tothe N-terminus of every copy of the major phage coat protein.These random peptides constitute the ‘active site’ of the land-scape phage and include up to 25% by weight of the phage andup to a half of its surface area (an extremely large percentage incomparison with natural proteins including antibodies) [11].A large mixture of such phages, displaying up to a billion vari-ous guest peptides, is known as a ‘landscape library’. From thislibrary, phages can be affinity selected for specificity to a certainantigen, therefore, functionally mimicking antibodies [13]. Thesephages can be successfully and conveniently made and aresecreted from the cell nearly free of intracellular components ina produce of approximately 20 mg/ml [11]. The surface densityof the phage-binding peptides is 300–400 m2/g, similar to themost effective known absorbents and catalysts [11], and with alarge number of potential-binding sites per phage, makes a mul-tivalency. Additional advantages of phages over antibodiesinclude the remarkable durability of the phage particle. It is

resistant to heat (up to 70oC) many organic solvents (such asacetonitrile), urea (up to 6M), acid, alkali and many otherstresses [11,14]. Purified phages can be stored indefinitely at mod-erate temperatures without losing infectivity [11]. Therefore,phages may be practical as alternative antibodies in numerousapplications including biosensors, affinity sorbents, hemostatics,etc. Many examples of uses of both lytic and filamentous phagesas probes for biological recognition in biosensors have beendocumented in the literature (reviewed recently in [15]). Bacter-iophages have a significant place in the phage-based recognitionof antibiotic-resistant pathogens (TABLES 1–3).

Immobilization of phages on sensor surfacesThe physical adsorption of the phage to the surface is probablythe simplest method of phage immobilization on the sensorsurface. This method was previously successfully employed forimmobilization of a wide range of biological elements [11] (andreferences therein). Self-assembled monolayers, comprisingchemisorbed elements, play an important role in immobilizingphages. It was reported that modifying surfaces with cysteine,followed by treatment with 2% glutaraldehyde, resulted in a37-fold improvement of bacteriophage attachment comparedwith physical adsorption [16]. Other researchers tried to immo-bilize phages by exploiting the high affinity of biotin to strepta-vidin. Petrenko and Vodyanoy obtained the analyte-bindingaffinity to phage compared well with one found for antibodiesisolated from a phage display library [12]. Gervais et al. reporteda 15-fold increase in the attachment densities of bacteriophagesover physical adsorption [17]. Tolba et al. used phage displaymethods to introduce affinity tags and cellulose-binding mod-ule in the head of T4 bacteriophages to provide a uniform-oriented immobilization on solid surfaces [18]. Phages boundvia mixed self-assembled monolayers of L-cysteine and 11-mer-captoundecanoic acid permitted both the recognition and dis-ruption of bacterial membranes [19]. The Langmuir–Blodgetttechnique couples precision of film thickness measurementswith a high degree of structural positioning control over themolecular architecture and has been firmly established in bio-sensor nanotechnology [20].

Wild-type phagesOne can make use of the biological infection cycle of phages,which includes binding to the host cell, penetration to injectDNA, synthesis and growth to create the phage progeny andrelease to lyse the cell and free the freshly produced phages(FIGURE 1). Known assays take advantage of different steps of theinfection cycle and include phage binding, phage typing, phageamplification, electrochemical and optoelectronic assays, recentlyreviewed by [2,3]. The assays described below have employedtechniques for typical phage-based detection of antibiotic-resistant bacteria.

Phage bindingIn the first step of infection by phages, the phage particleattaches to the host bacteria and the resulting complex can be

Review Sorokulova, Olsen & Vodyanoy

176 Expert Rev. Med. Devices 11(2), (2014)

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Table

1.Phage-baseddetectionsystemsforantibiotic-resistantbacteria.

Targets

Phage

Method

Medium

Assaytime

Detectionthresh

old

Ref.

Bacillusanthracis

gPhag

eam

plification

Growth

media

20h

Concentrated

culture

[40]

B.anthracis

gPhag

e/DNAamplification(qPC

R)

Growth

media

5h

102cfu/m

l[39]

B.anthracis

gPhag

ecomponen

ts,PlyG

capture

element,ATPassay

Growth

media

60m

100spores/ml

[47]

B.anthracis

gPhag

ecomponen

ts,PlyG

capture

element(dotblotassay)

Growth

media

3h

103spores/ml

[48]

B.anthracis

gPhag

ecomponen

ts,PlyG

capture

element

Growth

media

90m

103spores/ml

[49]

B.anthracis,spores

Filamentousf8/8

phage,

cloneJRB7

Phag

ebinding,magnetoelastic

sensors

Spore

suspen

sion

Realtime

102spores/ml

[29]

B.anthracis

Wb

Reporter

phage(luxA

B)

Blood

80m

103cfu/m

l[42]

Mycobacterium

tuberculosis

D29

Phag

eam

plification,MTTand

plaqueassays

Sputum

2days

100cfu/m

l[38]

M.tuberculosis

TM4derivative

Reporter

phage,FFlux

Culture

3–4

h8�

101–5�

105cfu/m

l[44]

M.tuberculosis

TM4derivative

Reporter

phage,fluorescent

microscopyorflow

cytometry

Culture

16–18h

37–67%

cells

fluoresced,

<100cfu/m

l

[46]

M.tuberculosis

TM4derivative

Reporter

phage,fluorescent

microscopy

Clinicalstrains

2–3

days

93–95%

cells

fluoresced

[45]

M.tuberculosis

phAE142

Reporter

phage,MGIT

960

Sputum

1–2

w103cfu/m

l[43]

Salm

onella

typhim

urium

Phag

esselectedfrom

landscapelibrary

f8/8

Phag

ebindingELISA

,precipitation

tests

Bacterialsuspension

2–3

h104cfu/m

l[26]

S.typhim

urium

Filamentousphage,

f8/8

cloneE2

Phag

ebinding,QCM,phage

monolayer

Bacterialsuspension

3.5

m102cells/m

l[24]

S.typhim

urium

Filamentousphage

f8/8,cloneE2

Phag

ebinding,spheroids

monolayers,QCM

Bacterialsuspension

6m

102cells/m

l[25]

S.typhim

urium

Filamentousphage,cloneE2

Phag


sensors

Culture

3m

103cfu/m

l[22]

S.typhim

urium

Filamentousphage,cloneE2

Phag


sensors

Fat-freemilk

3m

5�

103cfu/m

l[23]

Salm

onella

spp.

Felix

O-1

Phag

eam

plification

Culture

8–2

4h

3�

106cfu/m

l[34]

Salm

onella

spp.

Felix

O-1

Phag

eam

plification

Milk

24h

4�

104cfu/m

l[35]

m:Months;MGIT:Mycobacteriagrowth

indicatortube;

MRSA:Methicillin-resistantStap

hylococcusaureus;

MSSA:Methicillin-sensitive

Stap

hylococcusaureus;NR:Notreported

;QCM-D:Quartz

crystalmicrobalance

with

dissipationtracking;w:Weeks.

Bacteriophage biosensors for antibiotic-resistant bacteria Review

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detected by the number of methods. When thephage binds bacteria, the cell mass is increased andthis fact can be recognized by a gravimetric sensors(reviewed by [21]). The binding event can beregistered by many other techniques, includingELISA, magnetoelastic sensors, surface plasmon reso-nance (SPR), optoelectronic and electrochemicalmethods [2,3].

The landscape phage library probes that bindpreferentially Salmonella typhimurium cells comparedwith other Enterobacteriaceae has been used as substi-tute for antibodies. The clone E from the landscapelibrary f8/8 was used to demonstrate the rapid detec-tion of S. typhimurium in water suspensions, culture,vegetables and milk (TABLE 1) [22–27]. The phageworked well with different platforms includingELISA, quartz crystal microbalances (QCM) andmagnetoelastic sensor (TABLE 1). The sensitivity ofmagnetoelastic sensor increased by five-times when asensor size was reduced by a factor of two [22]. Bac-teria detection is realized by measuring the resonancefrequency change due to the change in mass as bac-teria are captured onto the sensor surface (FIGURE 2).

A capture of S. typhimurium by lytic phages PRD1,P22 and PR772 on glass surfaces was visualized by afluorescent microscopy and quantified using Image-Pro Plus statistical software [28]. In this research, thebacterial capture capability of phage-coated surfaceswas characterized for S. typhimurium. Binding of thephages to a solid surface impacted their capture effi-ciency and rate of host membrane disorder. Further-more, the size and shape of the bacteriophage andplacement of its specific binding proteins substantiallyinfluenced its bacterial capture ability in the immobi-lized condition. Immobilized phages were discoveredto rapture the membranes of bound bacteria and aretherefore suggested as a choice for antimicrobialsurfaces [28].

A phage-binding technique was also used inphage-coated magnetoelastic microbiosensors forreal-time detection of B. anthracis spores in sporesuspension [29]. Another clone (JRB7) of the fila-mentous phage f8/87 was utilized in this method.Phage was immobilized onto the surface of the sen-sors by physical adsorption. Authors reported real-time measurements with a detection threshold of102 spores/ml using 1 � 0.2 � 0.15 mm sensors inspore suspension in a range of concentration levelsof 5 � 101 – 5 � 108 spores/ml.

The lytic phage (bacteriophage 12600) was selectedfrom the commercial mixture of phages by incubationwith the S. aureus ATCC 12600 strain. Selectedphage infects wide spectrum of Staphylococcus isolates.A detection of S. aureus was carried out by the phage12600 as a biorecognition element with a SPR-basedT

able

1.Phage-baseddetectionsystemsforantibiotic-resistantbacteria(cont.).

Targets

Phage

Method

Medium

Assaytime

Detectionthresh

old

Ref.

S.typhim

urium

Felix

O-1

Phageamplification

Culture

4h

60cfu/m

l[36]

S.typhim

urium

Filamen

tousphage,cloneE2

Phagebinding,magnetoelastic

sensors

Milk,fresh

tomato

surfaces

30m

5�

102cfu/m

l[27]

S.typhim

urium

P22PhageTSP

Phagecomponents,Surface

plasm

onresonan

ce

Culture

30m

103cfu/m

l[15]

S.typhim

urium

PRD1,P22,PR772

Phagebinding,Opticaldetection

Culture

2–5m

5�

102cfu/m

l[28]

Staphylococcusaureus

12,600

Phagebinding,Surface

plasm

on

resonance

Culture

8m

104cfu/m

l[30]

S.au

reus

12,600

Phagebinding,Opticaldetection

Culture

2–5m

106cfu

[56]

S.au

reus

(MRSA)

12,600

Phagebinding,QCM-D,Combined

phage-antibody

Culture

12m

104cfu/m

l[31,51]

S.au

reus

(MRSA)

BP14

Phagebinding,Surface

plasm

on

resonance

Culture

20m

103cfu/m

l[33]

S.au

reus

(MRSA)

PhagecocktailTh

eKeyPath

MSSA/M

RSAtest

Phageamplification

Blood

16.9

hHigh

[37]

Streptococcuspneu

moniae

M13tg131,lyticenzymesLytA,

Cpl-1andPal

Phagecomponents,killassays

Clinicalisolates

5h

104

[57]

m:Months;MGIT:Mycobacteriagrowth

indicatortube;MRSA:Methicillin-resistantStaphylococcusau

reus;MSSA:Methicillin-sen

sitive

Staphylococcusaureus;

NR:Notreported;QCM-D:Quartzcrystalmicrobalance

with

dissipationtracking;w:Weeks.



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SPREETA sensor [30] or a high-resolution optical microscope sys-tem [31,32]. A 10-times lower detection limit, but an increasedassay time (20 min), was achieved by a specially selected lyticbacteriophage that authors claimed can specifically recognizemethicillin-resistant strains of S. aureus by a SPR [33].

Phage amplificationAt the end of the infection cycle, produced phages are escapedfrom the host resulting from cell lysis. A rise in the amountsof phages can hence be taken as a measure of the successfulinfections. By combining the target-specific phages with thesample and overseeing if the amount of phages increases theexistence, a target bacterium in a sample can be detected. Thistechnique, known as phage amplification, was initially intro-duced by Hirsh and Martin in 1983 to detect the presence ofSalmonella spp., first in pure culture [34] and then in milk [35].Following addition of Salmonella phage Felix-01 to the sample,the elevated number of replicated phages released into themedium was determined by HPLC. Researchers demonstrateda detection limit of 4 � 104 cfu/ml with a 24 h assay time.This initial phage amplification method is limited by thenecessity of a large quantity of target bacteria within the sam-ple and expense and complex nature of HPLC evaluation.Using a modified phage amplification technique, a much lowerdetection limit of 60 cfu/ml and a shorter assay time with thesame Salmonella phage Felix-01 has been achieved [36]. Anessential component of this assay is a strong virucidal agentthat inflicts no injury to the infected target bacteria.Ultimately, any producing plaques are resulted only frominfected target microorganisms. Contrary to the conventionalphage typing technique, which requires the target pathogento be at sufficient amounts to develop a bacterial lawn, thephage amplification assay can recognize target cells at far lowerlevels through amplifying the initial infection signal with sec-ondary infection of the included helper cells. Additionally, it isbeneficial to identify target bacteria from a sample that alsoincludes various other bacteria because the nonhost cells arenot affected by the phage and are outnumbered by the helpercells with regards to development of the bacterial lawn. More-over, incorporating an additional antibiotic before the regularphage amplification assay makes the detection of antibiotic-resistant pathogens possible [37]. Commercial diagnostic kitsbased on phage amplification techniques are availablefor detecting antibiotic resistance in M. tuberculosis and ofMRSA (TABLE 2).

Instead of depending on the production of visual plaquesfollowing phage amplification, different endpoint assays can beapplied for the recognition of host-released phages or phagecomponents [3]. Colorimetric phage-based assay for detectionof rifampin-resistant M. tuberculosis has utilized an optical end-point assay using the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), live versus deadstaining. The technique eliminated preparation of indicatorplates, reducing both reagent and labor needs. The multiwellsetup enabled semiautomation by using multichannel pipettes Table

2.Commercialphage-baseddevicesto

identify

antibiotic-resistantbacteria.

Devicename(Company)

Targetbacteria

Media

Test

method

Tim

eto

resu

ltClinical

sensitivity/

specificity

LoD

501knumber/

reference

KeyPathTM

MRSA

/MSSA

BloodCulture

Test

(MicroPhage,Inc.)

Stap

hylococcusau

reus

MRSA

,MSSA

Bloodculture

Bacteriophag

e

amplificationgrowth

5h

93.2/99.5%

6.0

�105cfu/m

lK102342

xTAG�Gastrointestinal

PathogenPanel(GPP)/

LuminexMolecular

Diagnostics,Inc.

S.typhim

urium,

bacteriophageMS2as

acontrol

Humanstool

Qualitative

nucleicacid

multiplextest

24h

100/98.4%

2.34�

105cfu/m

lK121894

FastPlaqueTBTM

(BIOTEC

Laboratories

Ltd)

Mycobacterium

tuberculosis

Sputum

Bacteriophag

e

amplificationand

growth

24h

87.5/96.9%

>20plaques

[53,58]

FastPlaque-Response

TM

(BIOTECLaboratoriesLtd)

Mycobacterium

tuberculosis,rifampicin

resistance

Sputum

Bacteriophag

e

amplification

2days

100/100%

>50plaques

[54]



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and assisted in the working with large quantities of samples [38].A real-time PCR as an endpoint assay in the phage amplifica-tion method was employed for detection of B. anthracis at con-centration of approximately 102 cfu/ml in <5 h [39].

The traditional phage amplification assay still remains in usewhen the number of tested bacteria is high, and only identifica-tion of microorganisms is required. The use of this simple assaywith gamma phage for identification of B. anthracis vegetative

cells was tested with 51 B. anthracis strains and 49 similar non-B. anthracis Bacillus species. Repeatability, day-to-day precisionand analyst-to-analyst precision were very high [40].

Wild-type intact phages possess useful properties for theirapplication in biosensors of antibiotic-resistant bacteria. They canprovide relatively short assay time, high sensitivity and desirablespecificity (small or large range of hosts). Intact phages demon-strated to be used as probes in biosensors. Some intact lytic phages

preserve infectivity upon drying and can beapplied as a coating to create an antimicro-bial surface. Wild-type intact phages, how-ever, suffer from certain drawbacks thatlimit their application in biosensor plat-form. Biosensor probe efficiency of intactphages depends on finding successful cloneor selection from a natural bacterial habi-tat. Intact phages are biologically activeand thus result in lysis of the host bacte-rium upon infection that would lead toloss of signal on a biosensor platform [15].In addition, intact phages have relativelylarge sizes, which limit their application asbioreceptors on particular sensor platformssuch as in the SPR and quartz crystalmicrobalance-based sensors where detec-tion signal is distance dependent.

Modified phagesSeveral limitations in use of wild-typeintact phages as bioprobes for detectionof antibiotic-resistant bacteria can beovercome by engineered and structurallymodified bacteriophages. The capability

Table 3. Bacteriophages against antibiotic-resistant bacteria.

Phage Host bacteria Specificity Reaction time Ref.

jAB2 Acinetobacter Acinetobacter baumannii M3237 5 m [59]

AP22 Acinetobacter 89 of 130 of A. baumannii strains 80 m [60]

fAB2 Acinetobacter Wide spectrum of A. baumannii strains 8 m [61]

HP1 Neisseria gonorrhoeae N. gonorrhoeae strains 6 h [62]

M1-M5 Neisseria meningitidis N. meningitides, 20 strains 15 h [63]

NCTC 11237 Group B Streptococcus GBS, groups A, C, G, and L 2–24 h [64]

0507-KN2-1 Klebsiella Specific to KN2 capsular polysaccharides 6 h [65]

KP34 Klebsiella 47.1% of extended-spectrum beta-lactamase-

positive versus 36% of extended-spectrum

beta-lactamase-negative strains

6 h [66]

Vrep-5 Vancomycin-resistant

EnterococcusBroad range of vancomycin-resistant

Enterococcus and non-resistant strains

24 h [67]

jMR11 Vancomycin-

intermediate/resistant

Staphylococcus aureus

Methicillin-resistant S. aureus and

vancomycin-resistant S. aureus and a

subset of vancomycin-intermediate S. aureus

6 h [68]

Bacteriumanchored bybound phages

Free phages havereleased by burstinginfected bacterium

Phage bound tothe gold surface

Gold-coated quartz piece

Figure 1. Bacterium lysis by phage attached to the gold surface.Reproduced with permission from [31] � Elsevier (2012).



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to change the DNA of microorganismshas opened the way for remarkable abilityof developing new detection systems forbiosensor applications. Pathogen detec-tion using engineered bacteriophage wasrecently reviewed by [2,3]. This sectionwill focus on the modified bacteriophagesused for detection of antibiotic-resistantbacteria.

Reporter phagesIn the reporter phage technology, DNAcarrying a reporter gene is placed into atarget bacterium by using a bacterio-phage. When the reporter gene has beenintroduced to the bacterium, it isexpressed, thus enabling bacterial cells tobe easily recognized. Given that bacterio-phages require host cells to reproduce,the reporter gene will not be expresseduntil the phage DNA has been insertedinto the host. Consequently, expression of the reporter gene‘reports’ the presence of the infected bacteria. Prokaryotic andeukaryotic luciferase-expressing gene (lux and luc), Escherichiacoli b-galactosidase (lacZ ) gene, bacterial ice nucleation (inaW )gene and green fluorescent protein-expressing gene have beenmost frequently used for such applications [2,41].

The reported phages used in biosensing are many and varied.A luxAB-tagged reporter phage was produced with the B.anthracis Wb temperate phage by replacing genes wp40 andwp41 with the luxAB genes encoding bacterial luciferase couldidentify approximately 103 CFU/ml vegetative cells with 80 minassay time [42]. The luciferase reporter phage, phAE142, derivedfrom a tempered mycobacteriophage L5, was used for detectionof antibiotic-resistant M. tuberculosis in sputum [43]. Muchhigher sensitivity and shorter assay time have been achieved byexpressing luciferase gene (FFlux) in both temperate (Che12)and lytic (TM4) phages for detection of M. tuberculosis [44–46].

Phage componentsBecause of the natural specificity of phages for their hosts,some of the phage components are also being used as detectionelements. Bacillus anthracis phage constituents are used as aprobe for detection. The PlyG lysin is utilized by the phage tohydrolyze peptidoglycan components, causing cell wall lysis andthe simultaneous release of progeny phages. If purified PlyG isadded externally to the cell, then fast lysis occurs leading to therelease of ATP [47]. When the enzyme luciferase and luciferinare added, an enzymatic reaction takes place leading to lightemission. Since PlyG reveals B. anthracis specificity, signalought to be created if only B. anthracis cells are present andthe lysin can trigger cell lysis leading to ATP release. That way,about 100 cells were detected with a hand-held luminometerwithin 60 min following addition of PlyG. Furthermore, thePlyG is utilized as a capture element for B. anthracis [48,49].

PlyG consists of two functional domains; the N-terminal cata-lytic domain and the C-terminal-binding domain that specifi-cally bind to the cell wall of B. anthracis. The binding domainof PlyG was fused to glutathione S-tranferase to produce arecombinant bioprobe able to binding and detecting B. anthra-cis. If utilized together with the horseradish peroxidase system,then about 103 cfu/ml were detectable within 3 h. Signifi-cantly, the PlyG could bind both nonencapsulated and encap-sulated bacterium, suggesting that it might have application forclinical samples. Another group of lytic enzymes obtained fromphages was demonstrating to target S. pneumonia.

Genetically engineered tailspike proteins originated fromphage P22, attached to gold-coated surfaces through cysteinetags at their N or C termini, could successfully work as molec-ular probes [15]. Utilizing SPR, the sensitivity of real-timedetection of S. typhimurium was 103 cfu/ml with 30 m ofdetection time.

SpheroidsGriffith and coworkers [50] demonstrated that filamentous bac-teriophages transformed into hollow spherical particles uponexposure to a chloroform–water interface. Filamentous phageaffinity selected for S. typhimurium through phage screen oflocal library had been converted with chloroform into circulartypes (spheroids) then transferred phage coating proteins mono-layers to QCM by Langmuir–Blodgett techniques to create bio-selective detectors [25].

A structurally altered lytic bacteriophage possessing a broadhost range of S. aureus strains and a penicillin-binding protein(PBP 2a) antibody conjugated latex beads have been employedto develop a biosensor made for discrimination of methicillin-resistant (MRSA) and sensitive (MSSA) S. aureus species [31,51].When bacteria are attached to the long flexible tails of the lyticphage, the distance between the attached cells and the sensor

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Figure 2. The wireless nature of the magnetoelastic biosensors and the basicprinciple for detecting bacterial cells. The fundamental resonant frequency of thebiosensor is fNo Mass without bacteria binding, which shifts (decreases) to fMass due tothe increased mass of bacteria binding to antibody immobilized on the sensor surface.Reproduced with permission from [55] � Elsevier (2007).



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surface is larger than the penetration of the QCM’s acousticwave; therefore, the signal of the binding event is not generated(FIGURE 3A). Replacing the intact phage with spheroids results infully functional phage with short tails, which bind the bacteriawithin the penetration depth of the QCM’s acoustic wave,therefore contributing to the frequency and dissipation changes(FIGURE 3B). When phage spheroids are deposited on a quartzcrystal microbalance with dissipation tracking (QCM-D) crystaland exposed to the mixed bacterial suspension, they bind boththe MRSA and MSSA strains of S. aureus but no other bacte-ria, separating the S. aureus from the other bacteria on the crys-tal surface (FIGURE 3C). Finally, when MRSA-specific, PBP2a-antibody-bound beads are added, a signal is generated in thepresence of MRSA strains of S. aureus (FIGURE 3D).

The lytic phages have been transformed into phage spheroidsby exposure to water–chloroform interface. Phage spheroidmonolayers have been transferred onto a biosensor surface byLangmuir–Blodgett method [52]. The produced biosensors havebeen evaluated by a QCM-D to assess bacteria–phage

interactions. Bacteria–spheroid interactions resulted in decreasedresonance frequency and a rise in dissipation energy for bothMRSA and MSSA strains. Following the bacterial binding,these biosensors have been additionally exposed to thepenicillin-binding protein antibody latex beads. Sensors exam-ined with MRSA reacted to PBP 2a antibody beads; however,sensors analyzed with MSSA gave no response. This unique dif-ference establishes an unambiguous discrimination betweenmethicillin resistant and sensitive S. aureus strains (FIGURE 4). Fur-thermore, bound and unbound bacteriophages control bacterialgrowth on surfaces and in water suspensions. After lytic phagesare turned into spheroids, they retain their strong lytic activityand possess high bacterial capture capability.

Commercial devicesThere are only a few commercial devices that detect antibiotic-resistant bacteria that utilize bacteriophage-based technology(TABLE 2). MRSA/MSSA blood culture test developed by Micro-Phage Inc. The device is allowed for prescription use in clinicsas a clinical aid in combination with other laboratory tests thatare utilized in the recognition and diagnosis of MRSA frominfected patients’ samples. The device is based on the bacterio-phage amplification method and has a low detection limit of6 � 105 cfu/ml with 5 h time to result. Two more commer-cialized devices using bacteriophage amplification were devel-oped by BIOTEC Laboratories, Ltd for detection and for testof antibiotic resistance of M. tuberculosis in sputum [53,54].

Another US FDA approved system, xTAG� Gastrointestinalpathogen panel (GPP) by Luminex Molecular Diagnostics(TABLE 2). The panel is intended for the simultaneous qualitativedetection and identification of multiple viral, parasitic and bac-terial nucleic acids in human stool specimens from individualswith signs and symptoms of infectious colitis or gastroenteritis.Multiple pathogens including S. typhimurium are included inthe panel. The method is based on the reverse transcription–polymerase chain reaction or RT-PCR/PCR and has a lowdetection limit of 2. 3 � 105 cfu/ml in 24 h for detection ofS. typhimurium.

Phages that recognize antibiotic-resistant bacteriaThere are a few antibiotic-resistant bacteria from the CDClist [1] that has counterpart bacteriophages, but phage-baseddetection assays have not yet been developed (TABLE 3). This factopens excellent opportunities for development of these assays inthe nearest future.

Expert commentaryAntibiotic-resistant bacteria became a serious problem for medi-cine and public health worldwide. Resistant strains of patho-gens are implicated in serious infection, nosocomial outbreaksand can be used as potential biowarfare agents. There is anurgent need in real time, sensitive methods of antibiotic-resistant pathogens’ detection. Conventional methods of patho-gen detection rely on the microbiological and biochemical testsbecause they are sensitive, specific and accurate. But these

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Figure 3. Short tail lytic phage spheroid and PBP 2A anti-body for discrimination of methicillin-resistant and-sensitive Staphylococcus aureus. (A) QCM-D crystal withintact phage. (B) The intact phage is replaced with spheroid. (C)Phage spheroids bind both MRSA and MSSA strains of Staphylo-coccus aureus, but do not bind other bacteria. (D) Suspension ofbeads with PBP 2a-specific antibody binds only MRSA thatcontain PBP 2a protein and do not bind MSSA.MRSA: Methicillin-resistant Staphylococcus aureus; MSSA:Methicillin-sensitive Staphylococcus aureus; PBP: Penicillin-bindingprotein; QCM-D: Quartz crystal microbalance with dissipationtracking.



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Figure 4. Combined quartz crystal microbalance with dissipation tracking and EM analysis of phage–bacteria interactions.1, 2 represent changes in the resonance frequency and the energy dissipation, respectively. (A) Phage-coated QCM-D sensor response toMRSA. Arrow shows the MRSA delivery time to the sensor surface. (B) Scanning electron micrograph of postassayed MRSA bound to lyticphage immobilized on the QCM sensor. M-MRSA, P-phages on the sensor surface. (C) Successive responses of the phage spheroids coatedQCM-D sensor to MRSA first and then to PBP antibody beads. Arrows indicate MRSA and PBP antibody delivery time to the sensor surface.(D) Scanning electron micrograph of postassayed biosensor with phage spheroids, MRSA and PBP antibody beads. Thick and thin arrowsshow typical MRSA cell and antibody bead, respectively. (E) Successive responses of the phage spheroids coated QCM-D sensor to S. aureusfirst and then to PBP antibody beads. Arrows indicate S. aureus and PBP antibody delivery time to the sensor surface, respectively. (F) Scan-ning electron micrograph of postassayed biosensor with phage spheroids, S. aureus and PBP antibody beads. Thick and thin arrows showtypical S. aureus cell and antibody bead, respectively.MRSA: Methicillin-resistant Staphylococcus aureus; PBP: Penicillin-binding protein; QCM-D: Quartz crystal microbalance with dissipationtracking; S. aureus: Staphylococcus aureus.Reproduced with permission from [31] � Elsevier (2012).



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methods are time-consuming and ineffective for on-site diagno-sis. More advanced methods for diagnosis of pathogens includePCR and ELISA. Bacteriophages are unique class of naturallyevolved recognition probes for bacteria. They are highly specificto host bacteria and stable in wide range of environmental con-ditions. Therefore, phages may be practical as alternative anti-bodies in numerous applications including biosensors, affinitysorbents, hemostatics, and so on. Bacteriophages were effec-tively used in different biosensors as recognition elements fordetection of antibiotic-resistant pathogens. Genetic and struc-tural modifications of bacteriophages have opened the way forremarkable ability of developing new detection systems for bio-sensor applications.

Five-year viewDevelopment of bacteriophage-based sensors is a very promis-ing approach for detection of antibiotic-resistant pathogens.Although great progress in this field has been achieved duringlast years, there are several problems to solve in the future.New technologies should be developed to decrease minimal

detection threshold of pathogens, because the infectious dose ofsome pathogenic bacteria is very low (e.g., 10 or less spores forB. anthracis). Detection time should be minimal, near realtime. This is of great importance for beginning of adequatetreatment and prevention of the pathogens’ spread. Develop-ment of new biosensors will be focused on the discriminationbetween antibiotic-resistant and -sensitive bacteria. Shelf life ofbiosensors should be extended. Scientific data indicate thatphage biosensors can be stable for several months and reusable.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with

any organization or entity with a financial interest in or financial conflict

with the subject matter or materials discussed in the manuscript. This

includes employment, consultancies, honoraria, stock ownership or options,

expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

The views expressed in this article are those of the authors, and do not

reflect the official policy or position of the United States Air Force, Depart-

ment of Defense, or the US Government.

Key issues

• Antibiotic-resistant bacteria are a serious problem for medicine and public health worldwide.

• Real-time, sensitive methods of detecting antibiotic-resistant pathogens are needed to start adequate therapy and prevent the spread

of infection.

• Current methods for diagnosis of pathogens are usually based on antibodies application. But cross-reactivity of polyclonal antibodies

and high production cost of monoclonal antibodies are limitation factors for these methods.

• Bacteriophages (phages) are viruses of bacteria. They are highly specific and can recognize only bacteria of one species or only one

particular strain. Specific bacteriophage can be isolated for any bacteria.

• Bacteriophages, in contrast to antibodies, are stable in wide range of environmental conditions.

• Efficacy of phages and their components as recognition elements on biosensor surface was shown in the detection of antibiotic-resistant

bacteria.

• Genetic and structural modifications of bacteriophages gave new opportunities for development of highly specific and selective probes

for detection of antibiotic-resistant bacteria.

• Phage can be used for developing of new technologies to decrease minimum detection threshold of pathogens.

• Phage biosensors can be stable for several months and reusable.

• Development of new biosensors will be focused on the discrimination between antibiotic-resistant and -sensitive bacteria.

References

Papers of special note have been highlighted as:

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Bacteriophage biosensors for antibiotic-resistant bacteria