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1. Introduction
Separation techniques are widely used for the analysis of biomolecules as well as small molecules in various fields, as genomics, proteomics or pharmaceutical sciences. Due to the wide range of separation techniques, numerous studies have been conducted aiming to improve performances in terms of sample preparation, sensitivity, cost or analysis throughput.
Liquid chromatography (LC) is the most employed separation technique, but alternative techniques such as capillary electrophoresis (CE) and gas chromatography (GC) are nevertheless helpful to provide orthogonal separation capabilities. Ultraviolet, electrochemical and fluorescence detection are used to detect the target compounds, but mass spectrometry (MS) detection offers enhanced sensitivity and additional structural information since co-eluting compounds are differentially detected according to their mass-to-charge ratio.
Miniaturisation is a general trend common to many areas in sciences and technology. Downscaling the separation techniques has been initiated in the 1970s, but miniaturisation has mainly experienced an exponential growth since the 1990s. Reducing the size of the separation supports brings valuable advantages as analysis time reduction, increased sensitivity and low sample and reagent consumption. However, the limited loading capacity of microfluidic devices is a drawback. Adequate sample preparation, pre-concentration and appropriate device can circumvent these inherent limitations.
2. Microfluidic separation techniques
2.1. Capillary electrophoresis
2.1.1. Instrumentation
Capillary electrophoresis (CE) is a microscale analytical technique based on the separation of compounds according to their charge-to-size ratio. The first CE device was described by Hjertén in 1967 that performed the electrophoretic separations in narrow bore tubes of 300 μm inner diameter (i.d.) for the analysis of various analytes (inorganic ions, nucleotides, proteins) [1]. In 1981, Jorgenson and Lukacs demonstrated for the first time that capillaries with a smaller i.d. (75 µm) could provide high separation efficiency using high voltages (30 kV), due to the small capillary dimensions that allowed good dissipation of Joule heat produced by such a high voltage [2].
Most modern CE instruments are very simple: a high voltage power supply, an autosampler with injection system, a capillary (25–100 µm i.d. and more often 50–75 µm, 20–100 cm length) and a detector coupled to a computer for data acquisition (Figure 1A).
Briefly, a capillary made of fused silica coated with a layer of polyimide is filled with a background electrolyte solution. When an optical detection (commonly UV or fluorescence) is employed, two electrodes are placed in buffer reservoirs to provide the necessary electrical contact between the high voltage supply and the capillary. To perform an analysis, the sample is loaded into the capillary by applying either a pressure difference (hydrodynamic injection) or an electric field (electrokinetic injection) between both extremities of the capillary. Optical detection is performed through a detection window directly on the capillary.
The hyphenation of CE to MS was first presented in 1987 by Olivares et al. that proposed an interface between CE and MS with an electrospray ionisation source (ESI) [3]. Alternative ionisation method has been described for CE-MS [4], as continuous flow-fast atom bombardment ionisation (CF-FAB) [5], atmospheric pressure chemical ionisation (APCI) [6] or atmospheric pressure photochemical ionisation (APPI) [7]. The CE-MS coupling provides structural information, enhanced sensitivity and selectivity compared with an optical detection. The CE-MS instrument configuration is modified to allow the direct entrance of the analytes into the mass spectrometer (Figure 1B).
Figure 1.
Schematic representation of a classical capillary electrophoresis system with an optical detection (A) and the coupling to a mass spectrometer (B).
The coupling of CE to MS can be achieved using a sheath liquid interface or a sheathless interface. The use of an additional liquid, the sheath liquid, to the electrophoretic effluent allows the formation of an electrical contact at the capillary MS output that is necessary for the electrophoretic separation, and enables the electrospray formation. In addition, the background electrolyte composition can be modified by dilution with the sheath liquid to ensure the compatibility with MS detection. However, the dilution process decreases the sensitivity in proportion with the sheath liquid flow rate.
Sheathless interfaces overcome the dilution-related sensitivity limitations encountered when using a sheath liquid interface. In this configuration, the electrical contact cannot be established through a liquid junction; the electrical contact may be established by many different techniques, e.g. by the insertion of the separation capillary into a conductive sprayer or the coating of the outlet end of the capillary by a conductive material.
2.1.2. Capillary electrophoresis on chip
In the light of the small dimensions of the separation capillary, classical CE is naturally classified into the category of miniaturised separation techniques. During the past few years, a new trend in CE instrumentation has emerged: the miniaturisation of CE into an integrated chip device for hyphenation to MS [12]. Since the introduction of the first chip-based electrophoresis device by Manz et al. [13], chip design has undergone continuous evolution from a single-channel design to more complex layouts integrating all the analytical steps on a single component. The actual classical chip design is made of two crossed microchannels, solution reservoirs for the sample and the waste, and reservoirs at the cathode and the anode for the buffer (Figure 2A) [14].
Interfacing a CE chip to an ESI-MS detector can be realised by spraying directly from the chip [15] or from a capillary sprayer attached to the chip [16] (Figure 2B).
Figure 2.
CE chip (A) and CE-MS (B) chip basic configuration.
In 1999 Agilent launched the Bioanalyser 2100, the first commercial microfluidics-based platform for DNA, RNA, protein and cell analysis. Separation is performed by capillary electrophoresis in channels containing a gel matrix. This device is the miniaturised counterpart of gel electrophoresis analysis (e.g. SDS-PAGE for proteins and agarose gel electrophoresis for nucleic acids) [17, 18]. In this device, sample with a volume between 1 and 6 µl moves through the loading channels, and a fraction of this volume is injected into the separation channel filled with a gel matrix. Fluorescence detection is performed on the chip itself. Total analysis time (including sample loading, separation, staining and destaining) is 30–40 min on the Bioanalyser chip, what is much shorter than the few hours (up to 1 day) required for the classical gel electrophoresis process.
Advantages of CE downscaling are reduced analysis times (minutes to seconds), low sample volume requirements (to the picolitre range), low solvent consumption and high throughput capabilities through the possibility of performing simultaneous separations in parallel channels [12].
Many applications using CE on chip have been developed for the analysis of a wide range of matrices and analytes: food analysis (including small molecules, organic acids, heavy metals, toxins, microorganisms or allergens) [19], amino acid analysis [20] or even intact protein characterisation [21].
2.2. Gas chromatography
Gas chromatography (GC) was first described by James and Martin in 1952. They presented a separation of volatile fatty acids on diatomaceous earth impregnated with a mixture of silicone oil and stearic acid as stationary phase, and a flow of nitrogen as the mobile phase [22]. GC underwent an explosive progression during the next decade, with applications for the petroleum industry [23], followed by biochemical applications [24, 25].
First GC separations were performed on packed columns of 1–5 mm i.d. Column length limitations due to backpressure drop led to the introduction of capillary GC columns [26, 27]. In such columns, the stationary phase is coated on the inner walls of the capillary to form a thin film (wall-coated open tubular, WCOT), or impregnated into a porous layer (porous layer open tubular, PLOT) [28]. Since capillary GC columns have less than 1 mm i.d. (typically 0.05–0.53 mm), this technique could already be considered as miniaturised.
However, since the 1970s researchers have been trying to integrate all the components of a gas chromatographer including the detector on a single piece, or chip. In 1979, Angell proposed a silicon wafer chip including a sample injection valve, a 1.5 m column and a detector [29] (Figure 3). In the next decades, other homemade chips were proposed, but to our knowledge no commercial version of a small and portable GC chip has been proposed so far.
A few applications have been developed on microbore GC systems, but research is still more dedicated to reliable miniaturised system development rather than method development [32].
2.3. Liquid chromatography
Liquid chromatography is the most commonly used separation technique with a wide range of applications. The precursor of liquid chromatography was the Russian scientific Mikhail Semenovich Tswett. He discovered that plant leave extracts poured on a column packed with particles could be separated into distinct coloured bands. In 1956, Van Deemter published his famous work about the fundamental equation of the relationship between mobile phase linear velocity and height equivalent to a theoretical plate (Figure 4) [33]. The modern appellation high pressure (now interchangeable with performance) liquid chromatography was first introduced by Horvath in 1970 to designate liquid chromatography performed on reduced (<10 µm) porous particles. Since the 1970s, LC underwent an explosive popularity to become a standard separation technique with continuous progress in stationary phase variety and performances, hardware features and fields of applications.
Figure 4.
Van Deemter plot deconvolution: (A) Eddy diffusion term; (B) longitudinal diffusion term; (C) resistance to mass transfer term.
Two major research axes of LC have been developed (Figure 5) to comply with the growing needs in increasing the analysis throughput, enhancing sensitivity and reducing analysis cost and environmental footprint through a decrease in solvent consumption [35].
2.3.1. Stationary phase particle size reduction
Considerable gains in terms of sensitivity and analysis time (or chromatographic resolution) could be obtained by reducing the stationary phase particle size to less than 2 µm, giving rise to ultra-high performance liquid chromatography (UHPLC). The use of smaller particles can significantly reduce the height equivalent of a theoretical plate (HETP) generated in a separation.
HETP=A+Bu+CuE1
\n\t\t\t\t\t
where u is the mobile phase velocity, A is the Eddy diffusion term, B is the longitudinal diffusion term and C is the resistance to mass transfer term.
The C term mobile phase component Cm can be expanded to the following relationship, showing its dependency on the square of particle size:
Cm=ωdp2DmE2
\n\t\t\t\t\t
where k is the retention factor, dp is the particle diameter, Dm is the diffusion coefficient of the solute in the mobile phase and ω is the pore size distribution, shape and particle size distribution coefficient.
Figure 5.
Historical trends in development of HPLC and on-chip LC (adapted from Lavrik et al.’s review [34]).
Particle size reduction has been initiated since the beginning of the spreading of HPLC as a separation technique, but technical limitations related to the pressure drop caused by particle size reduction delayed the commercialisation of sub-2 µm particle columns with classical dimensions [36]. The first pumps able to face ultra-high pressure were presented by Jorgenson [37] shortly followed by Lee et al. in the late 1990s. In 2004, Waters commercialised the first UHPLC system that was design to deliver pressure up to 1000 bar [41].
2.3.2. Column inner diameter reduction
In parallel with the reduction of particle size, the miniaturisation of LC columns in terms of inner diameter encounters a growing interest since the late 1970s [42].
As previously developed, downscaling the inner diameter of the separation support increases sensitivity with up to 3–4 orders of magnitude in a reduced analysis time. In addition to the advantages of UHPLC, LC miniaturisation reduces drastically the requirements in terms of sample and mobile phase volume.
Miniaturised columns operated on classical LC systems have been described, but void volumes that are very large compared with flow rates and column volumes are responsible for peak dispersion. For that reason, the integration of chromatographic components on a chip (separation channels and electrospray emitters for MS detection, but also additional channels, connections and microvalves) has rapidly been the major strategy to minimise void volumes and efficiency drop [43].
In 1978, Tsuda and Novotny experienced with the performances of packed glass capillaries with 50–200 µm inner diameters.
During the next years, research on chip technology was mainly focused on electroosmosis- or electrophoretic-driven separations due to the technical challenge represented by the connection between LC pumps and chips.
2.3.2.1. Open-channel chromatography
The simplest way to perform miniaturised liquid chromatography on chip is to coat the inner walls of the channels with chemical groups that may interact with the compounds of interest, i.e. to perform open-channel chromatography. In 1990, Manz et al. proposed the first chip prototype for open-tubular liquid chromatography made of silicon and coupled to a miniaturised conductometric detector connected to a classical LC pump [46]. Jacobson et al. proposed the first open-channel separation application on a glass chip coated with octadecylsilane chains in 1994, with low theoretical plate heights (4.1–5 µm) [47]. Due to the small specific surface of such systems, researchers conceived coating modifications to increase the phase ratio (ratio between the volume of stationary phase and the volume of mobile phase): porous layer open-tubular (PLOT) columns, functionalised particles embedded in a porous layer [50] or immobilisation of nanoparticles onto the walls [51].
Open-channel chromatography (with an ideal i.d. of 10–20 µm) provides high efficiency since the molecular diffusion is the only contributor to band broadening. However, due to its limited specific surface, column capacity stays low even with stationary phase modifications.
2.3.2.2. Micropillars, collocated monolith support structures and nanotubes
Micropillars, collocated monolith support structures (COMOSS) or nanotubes may combine small channel dimensions and large specific surfaces. COMOSS were introduced in 1998 by He and Regnier in response to the difficulty to produce chromatographic columns from wafers [52]. They proposed an approach where the stationary phase is not created by polymerisation in situ, but by etching the chip material (e.g. quartz, polydimethylsiloxane (PDMS) or cyclic olefin copolymer (COC)) that may be further functionalised. The result is a highly well-ordered structure (Figure 6) obtained as separation support. Eddy diffusion term in the Van Deemter equation is consequently much reduced, leading to high separation efficiency.
Figure 6.
SEM image of a COMOSS organised structure [52].
PDMS can be considered as a C1 phase, but its hydrophobicity is too low to perform adequate separation; PDMS monolithic pillars could therefore be functionalised by octasilane, octadecylsilane or other groups to improve analyte separation (Figure 7).
Figure 7.
PDMS functionalisation.
COC stationary phase has been presented for the first time by Gustafsson et al. in 2008 [55]. This material presents interesting features in terms of chemical inertness and stability in hydro-organic solvents. The hydrophobic character of COC allows it to be used as chip substrate and stationary phase. COMOSS chips made of on-porous materials as PDMS and COC have high separation efficiency, but low sample capacity due to the low interaction surface.
In addition to non-porous materials as PDMS or COC, superficially porous pillars have been proposed to circumvent the low sample capacity. Two orders of magnitude could be gained in terms of specific surface, increasing the chip sample capacity [56]. Another approach is the in situ growth of nanotubes on a COMOSS structure. Increased sample loading capacity and better retention than with C18-functionalised pillars could be obtained [43].
2.3.2.3. Miniaturised monolithic columns
Monoliths are continuous stationary phase beds generated by in situ polymerisation of monomers in the presence of porogen agents, resulting in a bimodal structure that exhibits macropores (>50–100 nm) that allow the mobile phase to pass through the column, and mesopores (<20 nm) that offers a high interaction surface for analyte retention [57]. Monolith retention properties can be defined before the polymerisation process by adjusting reagent nature and proportion, or by functionalising the polymer bed. Monoliths present undeniable chromatographic features and deserve to be more thoroughly understood in terms of synthesis parameters and their impact on chromatographic properties [43].
2.3.2.4. Packed particles
Besides the above-mentioned novel LC-chip stationary phases, silica particles can also be employed with the advantage of being well-known due to their broad utilisation for decades in classical LC; a wide range of particle functionalisation types and specifications have been commercialised for a long time. However, special attention has to be paid to particle packing homogeneity and immobilisation of the particles inside the microchannel.
Different column packing procedures have been developed to find the best way to obtain homogenous particle beds. Particles could be brought into chromatographic channels and trapped between weirs or frits that prevent further particle movements. Micromachined frits demonstrate better efficiency than sintered frits that generate more band dispersion [61]. Another procedure was developed for the first time in 2002 by Ceriotti et al. [62]. They proposed a fritless configuration where the particulate bed is retained in the chromatographic channel by a tapered profile at the end of the column. Improvements to this concept were proposed by Gomez et al. that presented a packing process with increased particulate bed stability.
2.3.3. The Agilent HPLC-chip
In 2005, Agilent developed and commercialised a miniaturised HPLC-chip system designed for direct coupling to a mass spectrometer [60]. Polyimide was chosen as chip substrate material due to its chemical and physical inertness, and the low MS background generated. The fabrication process consists in laser ablation of polyimide film to form the microfluidic channels, ports, chambers and columns followed by deposition of electrical contacts for the electrospray. The last step is the packing of the sample enrichment column and LC column with the stationary phase [65]. This latter operation is performed by introducing isopropanol particle slurries into both channels under a pressure of 120 bar. A wide range of particle chemistries, dimensions and porosities are available in classical Agilent LC columns that can be packed into the chip device.
Chromatographic separations on the Agilent HPLC chip are performed using pressure-driven mobile phase flow. Interfacing macrodimension pumps to nanodimension channels is made through a Chip-Cube interface in which the chip device is sandwiched between the rotor and stator (Figure 8) of a valve. Transfer capillaries from pumps, injector and to waste are connected to the valve stator, ensuring a tight and zero void-volume connection.
Figure 8.
Valve rotor (right) and stator (left) connected with transfer capillaries (A); cut-out view of fluidic connections (left), chip (middle) and valve rotor (right) [65].
Analysis on HPLC-chip consists of sample loading on an enrichment column pushed by a first pump equipped with a split flow device and working at capillary flow rate. After microvalve switching, a second pump delivering a split nanoflow rate is employed to perform chromatographic separation, passing through the enrichment column and the separation channel.
HPLC-chip hyphenation to MS is ensured by an electrospray emitter incorporated in the chip device. The electrospray tip is formed of a prolongation of the polyimide laminated films that constitute the chip substrate. The latter is laser ablated to the appropriate shape (45 µm diameter and 2 mm long) and coated with a conductive metal.
The integrated design of this miniaturised device reduces drastically void volumes and leakage possibilities. Moreover, HPLC-chip is easy to use and compatible with classical LC modules (pumps, autosampler/injector), which opened a wide field of applications.
Since its commercialisation in 2005, HPLC-chip has been used in qualitative analysis of tryptic peptides and proteins, and quantitative analysis of small molecules and peptides [72].
3. Interests of miniaturised LC
3.1. Injection volume
As in classical HPLC, the maximal volume that can be injected without causing a chromatographic band distortion is expressed by the following equation :
Vmax=θ.D.π.L.dc2.ϵc.(1+k)NE3
\n\t\t\t\t
where θ is the fractional loss of the column plate number caused by the injection, D is the constant describing the injection profile, L is the column length, dc is the column i.d., ∊c the is column porosity, k the is retention factor and N the is column efficiency expressed by the theoretical plate number.
As shown in this equation, Vmax is the proportional to the square of dc, and the following relationship can be established:
For two columns that have the same length, efficiency and porosity but differ by their inner diameter (4.6 mm for classical dimensions and 75 µm for the miniaturised version), a theoretical injection volume reduction factor of 3762 should be observed (e.g. 10 µl onto a conventional system to approximately 2.5 nl on a nano-LC column) while keeping the same chromatographic performances. Such a reduction of the required injection volume represents an undeniable advantage of miniaturised LC systems, since a growing interest is brought, for instance, to the analysis of biological matrices that are often available in limited volumes.
In practice, a great sensitivity gain can be obtained by injecting higher volumes onto the miniaturised chromatographic system, without causing peak distortion due to an overload. In the case of micro-LC, different peak compression techniques have been studied, such as on-column concentration or sample plug bracketing. In nano-LC, a trapping column is often connected to the analytical column by a valve, allowing large sample volumes to be loaded onto the system and the sample to be pre-concentrated.
3.2. Peak concentration
A reduction of the inner diameter of a chromatographic column results in a higher peak concentration at the detector (Cmax), as shown in the following equation:
Cmax=N2π×4mπ.L.Vo.ϵc.(1+k)E5
\n\t\t\t\t
where m is the total amount of sample loaded on the column and V0 is the column volume.
Cmax is the proportional to m and to N, and inversely proportional to V0. Since V0 is directly related to dc, Cmax is inversely proportional to the square of column diameter. In other words, Eq. (6) can be used to illustrate the sensitivity gain that can be expected with miniaturised columns.
Downscaling the size from classical dimensions (4.6 mm) to miniaturised dimensions (75 µm) would theoretically result in a gain factor of Cmax of 3762.
3.3. Void volume reduction
Void volumes are detrimental to the chromatographic performances in all LC configurations. However, when working with miniaturised systems, the smallest void volume can act as a mixing chamber and result in an important loss in sensitivity and separation efficiency. The total band dispersion occurring in a chromatographic system (Figure 9) can be expressed by the total variance σtot2 that sums the variances due to the column (σcol2) and to the rest of the chromatographic system (σext2).
σtot2=σext2+σcol2E7
\n\t\t\t\t
Figure 9.
Schematic representation of band broadening components in a chromatographic system (adapted from Lauer [79]).
Band dispersion due to the column, σcol2, is in particular a function of column volume (and consequently to dc2) and efficiency, which are physical properties that cannot be changed for a given column in order to decrease peak broadening:
σcol2=π.L.dc2.ϵc.(1+k)4NE8
\n\t\t\t\t
However, other factors having an influence on band broadening through σext2 can be expressed as:
σext2=Vinj2dc2+σ02E9
\n\t\t\t\t
where Vinj2 is the injection volume, σ02 is the instrument variance and σext2 is the extra-column variance.
As shown in this equation, Vinj2 and σ02 are directly related to σext2 [79]. In other words, the minimisation of extra-column void volumes by using the smallest connection capillaries and fittings possible is clearly beneficial to avoid chromatographic band dispersion.
In the light of these considerations, systems with very low extra-column void volumes have been developed including integrated systems (see Section 2.3.3).
3.4. Low flow rate
Mobile phase flow rate F is a value that is also related to the internal column diameter as seen in Eq. (10):
F=π.dc2.ϵc.u4E10
\n\t\t\t\t
where u is the mobile phase velocity.
The following relationship can be written in Eq. (11):
This drastic flow rate reduction has evident economical and ecological advantages, especially when working with pumping systems that directly deliver the right mobile phase flow rate without involving the use of a split flow system.
3.5. Retention volume
The retention volume VR is defined as the mobile phase volume that is required to elute a compound of a given retention time tR :
VR=tR.F=tR.π.dc24.ϵc.uE12
\n\t\t\t\t
In the light of the reduced column dimensions in miniaturised LC systems compared with classical systems, the mobile phase volume that is needed to elute a compound with a specified k value is reduced proportionally to the square of the internal column diameter, as shown in Eq. (13).
When using mass spectrometry, compounds of interest have to carry a net positive or negative charge, depending on the mode that is employed. Analyte electrospray ionisation occurs in three major steps: first, charged droplets are formed from the chromatographic eluent under the action of a strong electric field. The eluent takes the shape of a cone (the Taylor cone) when a critical electric field threshold is reached. A pneumatic assistance is required to provide stable droplet formation in the classical LC [80]. Then, charged droplets undergo Coulomb fission into smaller daughter droplets: eluent solvent progressively evaporates in the heated source until reaching the Rayleigh limit where the electrostatic repulsion forces are exactly equal to the surface tension of the solvent [81]. Beyond the Rayleigh limit, droplets become unstable and divide into smaller droplets. Eq. (14) presents the relationship between droplet charge and Rayleigh radius.
Q2=64π2ε0γRR3E14
\n\t\t\t\t
where Q is the droplet charge ,ε0 is the vacuum permittivity and RR is the Rayleigh radius.
The ion transfer from small droplets to the gas phase can happen following two mechanisms. The ion evaporation model described by Iribarne and Thomson is commonly admitted to describe the small ion formation [81]. According to this model, the electric field at the droplet surface becomes strong enough at an intermediate state and before reaching the Rayleigh limit to directly desorb ions from the droplet (Figure 10) [82].
Figure 10.
Ion evaporation model.
A second model proposed by Dole, or the charged residue model, could be appropriate to describe protein ionisation. This model suggests that successive Coulomb fissions occurring when the Rayleigh limit is reached, finally yielding droplets containing one single charge (Figure 11) [83].
Figure 11.
Charge residue model.
Nanoelectrospray (nano-ESI) source was first introduced in 1994 as a response to the development of low flow separation devices. Typical flow rates in nano-ESI are 200–1000 nl/min and the i.d. of spray emitter is about 10–20 µm. The interest of such a miniaturised ionisation source is the improvement of the overall ionisation efficiency (the number of ions recorded at the detector divided by the number of analyte molecule sprayed) [86]. Since signal intensity with ESI sources is concentration sensitive rather than mass sensitive, low analyte amount are advantageously detected at lower flow rates with higher peak concentrations thanks to the miniaturised technique, as previously explained. Lower flow rates as well as narrower emitter tip orifice produce smaller droplets (2–3 orders of magnitude reduction), and desolvation efficiency is increased: smaller initial droplet size requires less Coulomb fission and solvent evaporation to release charged compounds into gas phase, making a larger portion of ions available to detection.
4. Sample preparation
In the light of the previously described features of miniaturised separation techniques, having low volume samples with the highest concentration possible is a clear objective. On the other hand, analysis of complex media (e.g. environmental, forensic, food, pharmaceutical or biological samples) requires preliminary purification to isolate analyte from contaminants and interferences, and to avoid column or capillary blockage, reduced separation phase lifetime and MS ion suppression. In addition, sample preparation may allow analyte concentration and analyte matrix simplification to make the sample fully compatible with separation technique and detection.
The combination of miniaturised sample preparation and separation techniques offers the main advantages of high throughput, high sensitivity and low costs. The most employed miniaturised sample preparation techniques are briefly described below.
Liquid-liquid extraction (LLE) is a sample preparation technique that relies on the partition of analytes between two immiscible liquid phases. The best results are obtained for compounds showing a clear preference for one liquid over the other one. Factors that influence compound partition include liquid phase polarity, pH, analyte pKa and polarity, and mixing and contact duration. Micro liquid-liquid extraction (MLLE) is a simple downscaling of classical LLE procedure. The use of lower sample volumes has economical and ecological advantages since the apolar liquid phase is often constituted of alkanes (e.g. pentane, hexane and cyclohexane) or chlorinated solvents; moreover, reduced solvent volumes may lead to the increased analyte concentration.
Solid-phase microextraction (SPME) is a miniaturised sample preparation process involving a fused-silica rod coated with a polymeric layer employed as extraction medium. This technique is applied for the extraction of trace compounds from liquid or gas samples [94] (Figure 12). Analyte desorption is performed by heating the SPME fibre in a classical GC injector for volatile and thermally stable compounds, or by a special desorption device for non-volatile or thermally unstable compounds for subsequent LC [95, 96] or CE [97] analysis.
Figure 12.
Extraction from aqueous sample solution by conventional SPME device. (A) Liquid phase sampling and (B) headspace sampling [99].
Dried spots are an expanding way of microsampling and purifying biological samples as blood (dried blood spots, DBS), serum (dried serum spots, DSS) or plasma (dried plasma spots, DPS). A few microliters of a biological fluid are collected on a filter paper and allowed to dry. The dried spot is then punched out and desorbed in an appropriate mixture of solvent chosen to enable maximal analyte extraction while minimising interference desorption (Figure 13). In addition to analytical advantages as small sample volume requirements and low cost, dried spots are very convenient from a sampling point of view: the collection technique is not invasive and can be performed without pain, e.g. for pharmacokinetic studies on laboratory animals or for systematic disease screening on newborns.
Figure 13.
DBS sampling and extraction procedure.
Finally, solid-phase extraction (SPE) follows the miniaturisation trend by reducing cartridge and solid phase bed volume (Figure 14A and B). In this technique, sample is loaded in a tube containing a few mg to a few tens mg particles maintained in the bottom of the cartridge by two frits. Sample loading solvent has to be carefully chosen to ensure analyte retention on the particles. Washing steps are then performed to remove a maximal amount of contaminants and interferences that are co-retained on the solid phase, while maintaining analyte-particle interactions. Elution is the final step of SPE to collect a sample containing the analyte for further analysis. Downscaling SPE support allows preparing sample volumes as low as 10 µl, and analyte elution by similar volumes. Moreover, SPE or micro-SPE supports are increasingly available in 96-well format (Figure 14C) to provide high extraction throughput by the use of multichannel pipettes or extraction automation.
To summarise, the advantages of microfluidic devices include their small size, improved sensitivity, low sample volume requirements, rapid analysis, potential disposability, and importantly their ease of use that eliminates the need for skilled personnel to perform the assays. In the same time, ethical, analytical and sample availability considerations are a challenge faced by many (bio)analytical laboratories and have resulted in a drive to limit sample volume.
Integration of various nanotechniques through microfabrication processes and advances in detection devices and informatics drive new types of analysis facilitating on-site multicomponent analysis resulting in rapid diagnostic tools and rapid screening methods in various application fields (clinical, pharmaceutical and biopharmaceutical, environmental, food analysis, etc.).
Acknowledgments
The authors thank the Fund for Scientific Research (F.R.S.-FNRS, Belgium), the Walloon Region (WR), the Leon Fredericq Fund and University of Liege for financial support.
\n',keywords:"Separation, miniaturisation, microfluidics, sensitivity",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/52333.pdf",chapterXML:"https://mts.intechopen.com/source/xml/52333.xml",downloadPdfUrl:"/chapter/pdf-download/52333",previewPdfUrl:"/chapter/pdf-preview/52333",totalDownloads:1212,totalViews:331,totalCrossrefCites:1,totalDimensionsCites:1,hasAltmetrics:0,dateSubmitted:"May 29th 2015",dateReviewed:"July 19th 2016",datePrePublished:null,datePublished:"November 23rd 2016",dateFinished:null,readingETA:"0",abstract:"During the last decades, a great interest has been shown for miniaturised separation techniques. The use of microfluidic techniques fulfills the constant needs for increasing sample throughput and analysis sensitivity, while reducing costs and sample volume consumption. In this chapter, three microfluidic separation techniques will be addressed: capillary electrophoresis, gas chromatography and liquid chromatography. A special attention will be paid to miniaturised liquid chromatography, with a deep investigation of its advantages compared with classical liquid chromatography. Sample preparation adapted to low volumes (a few µl) will also be discussed.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/52333",risUrl:"/chapter/ris/52333",book:{slug:"advances-in-microfluidics-new-applications-in-biology-energy-and-materials-sciences"},signatures:"Virginie Houbart and Marianne Fillet",authors:[{id:"177056",title:"Prof.",name:"Marianne",middleName:null,surname:"Fillet",fullName:"Marianne Fillet",slug:"marianne-fillet",email:"marianne.fillet@ulg.ac.be",position:null,institution:null}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Microfluidic separation techniques",level:"1"},{id:"sec_2_2",title:"2.1. Capillary electrophoresis",level:"2"},{id:"sec_2_3",title:"2.1.1. Instrumentation",level:"3"},{id:"sec_3_3",title:"2.1.2. Capillary electrophoresis on chip",level:"3"},{id:"sec_5_2",title:"2.2. Gas chromatography",level:"2"},{id:"sec_6_2",title:"2.3. Liquid chromatography",level:"2"},{id:"sec_6_3",title:"2.3.1. Stationary phase particle size reduction",level:"3"},{id:"sec_7_3",title:"2.3.2. Column inner diameter reduction",level:"3"},{id:"sec_7_4",title:"2.3.2.1. Open-channel chromatography",level:"4"},{id:"sec_8_4",title:"2.3.2.2. Micropillars, collocated monolith support structures and nanotubes",level:"4"},{id:"sec_9_4",title:"2.3.2.3. Miniaturised monolithic columns",level:"4"},{id:"sec_10_4",title:"2.3.2.4. Packed particles",level:"4"},{id:"sec_12_3",title:"2.3.3. The Agilent HPLC-chip",level:"3"},{id:"sec_15",title:"3. Interests of miniaturised LC",level:"1"},{id:"sec_15_2",title:"3.1. Injection volume",level:"2"},{id:"sec_16_2",title:"3.2. Peak concentration",level:"2"},{id:"sec_17_2",title:"3.3. Void volume reduction",level:"2"},{id:"sec_18_2",title:"3.4. Low flow rate",level:"2"},{id:"sec_19_2",title:"3.5. Retention volume",level:"2"},{id:"sec_20_2",title:"3.6. Hyphenation to MS",level:"2"},{id:"sec_22",title:"4. Sample preparation",level:"1"},{id:"sec_23",title:"5. Conclusions and perspectives",level:"1"},{id:"sec_24",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Hjertén S. Free zone electrophoresis. Chromatographic Reviews. 1967;9:122–219. doi:10.1016/0009-5907(67)80003-6.'},{id:"B2",body:'Jorgenson JW, Lukacs KD. Zone electrophoresis in open-tubular glass capillaries. Analytical Chemistry. 1981;53:1298–302. doi:10.1021/ac00231a037.'},{id:"B3",body:'Olivares JA, Nguyen NT, Yonker CR, Smith RD. On-line mass spectrometric detection for capillary zone electrophoresis. Analytical Chemistry. 1987;59:1230–2. doi:10.1021/ac00135a034.'},{id:"B4",body:'Hommerson P, Khan AM, de Jong GJ, Somsen GW. Ionization techniques in capillary electrophoresis-mass spectrometry: principles, design, and application. 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Large injection volumes in capillary liquid chromatography: Study of the effect of focusing on chromatographic performance. Journal of Chromatography A. 2010;1217:7507–13. doi:10.1016/j.chroma.2010.09.076.'},{id:"B77",body:'Tao D, Zhang L, Shan Y, Liang Z, Zhang Y. Recent advances in micro-scale and nano-scale high-performance liquid-phase chromatography for proteome research. Analytical and Bioanalytical Chemistry. 2011;399:229–41. doi:10.1007/s00216-010-3946-7.'},{id:"B78",body:'Lauer HH, Rozing GP. The selection of optimal conditions in HPLC II. The influence of column dimensions and sample size on solute detection. Chromatographia. 1982;15:409–13. doi:10.1007/BF02261599.'},{id:"B79",body:'Lauer HH, Rozing GP. The selection of optimum conditions in HPLC I. The determination of external band spreading in LC instruments. Chromatographia. 1981;14:641–7. doi:10.1007/BF02291104.'},{id:"B80",body:'Ikonomou MG, Blades AT, Kebarle P. Electrospray-ion spray: a comparison of mechanisms and performance. Analytical Chemistry. 1991;63:1989–98. doi:10.1021/ac00018a017.'},{id:"B81",body:'Kebarle P. A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry. Journal of Mass Spectrometry. 2000;35:804–17. doi:10.1002/1096-9888(200007)35:7<804::AID-JMS22>3.0.CO;2-Q.'},{id:"B82",body:'Iribarne JV, Thomson BA. On the evaporation of small ions from charged droplets. The Journal of Chemical Physics. 1976;64:2287–94.'},{id:"B83",body:'Dole M, Mack LL, Hines RL, Mobley RC, Ferguson LD, Alice MB. Molecular beams of macroions. The Journal of Chemical Physics. 1968;49:2240–9.'},{id:"B84",body:'Wilm MS, Mann M. Electrospray and Taylor-Cone theory, Dole’s beam of macromolecules at last? International Journal of Mass Spectrometry and Ion Processes. 1994;136:167–80.'},{id:"B85",body:'Emmett MR, Caprioli RM. Micro-electrospray mass spectrometry: ultra-high-sensitivity analysis of peptides and proteins. Journal of the American Society for Mass Spectrometry. 1994;5:605–13. doi:10.1016/1044-0305(94)85001-1.'},{id:"B86",body:'Wilm M, Mann M. Analytical properties of the nanoelectrospray ion source. Analytical Chemistry. 1996;68:1–8.'},{id:"B87",body:'El-Faramawy A, Siu KW, Thomson BA. Efficiency of nano-electrospray ionization. Journal of the American Society for Mass Spectrometry. 2005;16:1702–7. doi:10.1016/j.jasms.2005.06.011.'},{id:"B88",body:'Abian J, Oosterkamp AJ, Gelpí E. Comparison of conventional, narrow-bore and capillary liquid chromatography/mass spectrometry for electrospray ionization mass spectrometry: practical considerations. Journal of Mass Spectrometry. 1999;34:244–54. doi:10.1002/(SICI)1096-9888(199904)34:4<244::AID-JMS775>3.0.CO;2-0.'},{id:"B89",body:'Juraschek R, Dülcks T, Karas M. Nanoelectrospray—more than just a minimized-flow electrospray ionization source. Journal of the American Society for Mass Spectrometry. 1999;10:300–8. doi:10.1016/S1044-0305(98)00157-3.'},{id:"B90",body:'Karas M, Bahr U, Dülcks T. Nano-electrospray ionization mass spectrometry: addressing analytical problems beyond routine. Fresenius\' Journal of Analytical Chemistry. 2000;366:669–76.'},{id:"B91",body:'Schmidt A, Karas M, Dülcks T. Effect of different solution flow rates on analyte ion signals in nano-ESI MS, or: when does ESI turn into nano-ESI? Journal of the American Society for Mass Spectrometry. 2003;14:492–500.'},{id:"B92",body:'Zapf A, Heyer R, Stan H-J. Rapid micro liquid-liquid extraction method for trace analysis of organic contaminants in drinking water. Journal of Chromatography A. 1995;694:453–61. doi:10.1016/0021-9673(94)01199-O.'},{id:"B93",body:'Montesinos I, Gallego M. Solvent-minimized extraction for determining halonitromethanes and trihalomethanes in water. Journal of Chromatography A. 2012;1248:1–8. doi:10.1016/j.chroma.2012.05.067.'},{id:"B94",body:'Louch D, Motlagh S, Pawliszyn J. Dynamics of organic compound extraction from water using liquid-coated fused silica fibers. Analytical Chemistry. 1992;64:1187–99.'},{id:"B95",body:'Salleh SH, Saito Y, Jinno K. An approach to solventless sample preparation procedure for pesticides analysis using solid phase microextraction/supercritical fluid extraction technique. Analytica Chimica Acta. 2000;418:69–77.'},{id:"B96",body:'Jinno K, Kawazoe M, Hayashida M. Solid-phase microextraction coupled with microcolumn liquid chromatography for the analysis of amitriptyline in human urine. Chromatographia. 2000;52:309–13.'},{id:"B97",body:'Whang CW, Pawliszyn J. Solid phase microextraction coupled to capillary electrophoresis. Analytical Communications. 1998;35:353–6.'},{id:"B98",body:'Saito Y, Kawazoe M, Imaizumi M, Morishima Y, Nakao Y, Hatano K, et al. Miniaturized sample preparation and separation methods for environmental and drug analyses. Analytical Sciences. 2002;18:7–17.'},{id:"B99",body:'Saito Y, Jinno K. Miniaturized sample preparation combined with liquid phase separations. Journal of Chromatography A. 2003;1000:53–67. doi:10.1016/S0021-9673(03)00307-8.'},{id:"B100",body:'Deglon J, Thomas A, Daali Y, Lauer E, Samer C, Desmeules J, et al. Automated system for on-line desorption of dried blood spots applied to LC/MS/MS pharmacokinetic study of flurbiprofen and its metabolite. Journal of Pharmaceutical and Biomedical Analysis. 2011;54:359–67. doi:10.1016/J.Jpba.2010.08.032.'},{id:"B101",body:'Abu-Rabie P, Spooner N. Dried matrix spot direct analysis: evaluating the robustness of a direct elution technique for use in quantitative bioanalysis. Bioanalysis. 2011;3:2769–81. doi:10.4155/bio.11.270.'},{id:"B102",body:'McDade TWS, Snodgrass J. What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography. 2007;44:899-925.'},{id:"B103",body:'Li W-T. Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules. Biomed Chromatogr. 2010;24:49-65.'},{id:"B104",body:'Britz-McKibbin P. Expanded newborn screening of inborn errors of metabolism by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). Methods Mol Biol. 2013;919:43-56.'},{id:"B105",body:'Gilar M, Bouvier ES, Compton BJ. Advances in sample preparation in electromigration, chromatographic and mass spectrometric separation methods. Journal of Chromatography A. 2001;909:111–35. doi:10.1016/S0021-9673(00)01108-0.'},{id:"B106",body:'Ekström S, Wallman L, Hök D, Marko-Varga G, Laurell T. Miniaturized solid-phase extraction and sample preparation for MALDI MS using a microfabricated integrated selective enrichment target. Journal of Proteome Research. 2006;5:1071-81. doi:10.1021/pr050434z.'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Virginie Houbart",address:null,affiliation:'
Laboratory for the Analysis of Medicines (LAM), Department of Pharmacy, CIRM, University of Liege, Liege, Belgium
Laboratory for the Analysis of Medicines (LAM), Department of Pharmacy, CIRM, University of Liege, Liege, Belgium
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Hill",authors:[{id:"79259",title:"Dr.",name:"Manh",middleName:null,surname:"Hoang",fullName:"Manh Hoang",slug:"manh-hoang"},{id:"86472",title:"Dr.",name:"Anita J.",middleName:null,surname:"Hill",fullName:"Anita J. Hill",slug:"anita-j.-hill"}]},{id:"35162",title:"Evaluation of Replacing Natural Gas Heat Plant with a Biomass Heat Plant - A Technical Review of Greenhouse Gas Emission Trade-Offs",slug:"evaluation-of-replacing-natural-gas-heat-plant-with-a-biomass-heat-plant",signatures:"James G. Droppo and Xiao-Ying Yu",authors:[{id:"24996",title:"Dr.",name:"Xiao-Ying",middleName:null,surname:"Yu",fullName:"Xiao-Ying Yu",slug:"xiao-ying-yu"},{id:"113768",title:"Dr.",name:"James",middleName:null,surname:"Droppo",fullName:"James Droppo",slug:"james-droppo"}]}]}]},onlineFirst:{chapter:{type:"chapter",id:"69255",title:"The War between Bacteria and Bacteriophages",doi:"10.5772/intechopen.87247",slug:"the-war-between-bacteria-and-bacteriophages",body:'
1. Introduction
Antimicrobial resistance is a global public health crisis. According to Public Health England [1], each year approximately 25,000 people die across Europe due to hospital-acquired infections caused by antibiotic-resistant and MDR bacteria such as Mycobacterium tuberculosis, Methicillin-resistant Staphylococcus aureus and multiresistant Gram-negative bacteria. Gram-negative infections include those caused by Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa [2]. Nevertheless, it is estimated that by 2050, the global yearly death toll will increase to 10 million. Accelerating emerge of antimicrobial resistance seriously threatens the effectiveness of treatments for pneumonia, meningitis and tuberculosis, in addition to diminishing prevention of infections acquired during surgeries and chemotherapies. The crisis of the antibiotic resistance requires urgent, coordinated action. Misuse and overuse of antibiotics must be controlled, implementation of new policies regarding prescriptions has to be internationally addressed; and development of new therapeutics is urgently required [1].
Félix d’Herelle, known as the father of bacteriophage (or phage) therapy [3], brought an evolutionary discovery of phages as therapeutics for various infections and conditions. Phage therapy was widely enforced in the 1920s and 1930s to combat the bacterial infections. However, in the 1940s, the newly discovered antibiotics replaced the phage therapy (except Russia, Georgia and Poland) [4].
The emergence of MDR bacteria prompted a renewal of the interest to the phage therapy as an alternative treatment to overcome a broad spectrum of resistant bacterial infections. Phage therapy and phage cocktails that contain a mixture of different bacteria-specific phages, drawn interest within molecular biology and modern medical research as potential antimicrobials that could tackle the crisis of antimicrobial resistance. Nonetheless, the phage therapy remains controversial due to its disadvantages such as bacteriophage resistance: bacteria-phage evolutionary arms race that could put a burden on a long-time application of phage therapy as an anti-infectious agent [5].
Phage therapy has many advantages, primary because phages are very specific (generally limited to one species) and easy to obtain as they are widely distributed in locations populated by bacterial hosts including soil and seawater, and they do not have any known chemical side effects like antimicrobials [6].
Understanding host-phage interactions and ‘the war between bacteria and phages’ are steps towards designing engineering ‘broad-spectrum phage’ that can overcome the limitations of phage therapy and potentially overcome a wide range of resistant bacterial infections [6].
2. The evolutionary phage-host arms race
Phages are obligate intracellular parasites that distinctively infect bacterial cells. Although phages are very specific to their host, generally limited to one species, they pose an enormous threat to bacteria as in some habitats they outnumber their hosts by nearly 10-fold number [7]. Phages are the most abundant, ubiquitous and diversified organisms in the biosphere [8, 9]. Phage-host interaction and fight for the survival led to the evolution of bacterial and viral genomes and, therefore, to the evolution of resistance mechanisms. Bacteria, continuously, evolve many molecular mechanisms, driven by gene expression to prevent phage infection. These evolving phage-resistance mechanisms in bacteria induce the parallel co-evolution of phage diversity and adaptability [10, 11]. The co-evolving genetic variations and counteradaptations, in bacteria and phages, drive the evolutionary phage-host arm race [11, 12].
Leigh Van Valen, an evolutionary biologist, metaphorised the co-evolutionary arm race and proposed the Red Queen hypothesis [13].
‘It takes all the running you can do, to stay in the same place’ the Red Queen says to Alice in Through the Looking-Glass.
The Red Queen hypothesis proposes that to survive, microorganisms must constantly adapt, evolve and thrive against ever-evolving antagonistic microorganisms within the same ecological niche [14].
Bacteria have developed various anti-phage mechanisms including non-adaptive defences (non-specific) and adaptive defences associated with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) along with CRISPR-associated (Cas) proteins [7, 15, 16, 17, 18].
The non-specific adaptations (analogues to innate immunity in multicellular organisms) act as primary mechanisms to evade viral infection, and they include mechanisms that inhibit phage adsorption and prevent nucleic acid entry, superinfection exclusion systems, restriction-modification systems and abortive infection [7, 19].
On the other hand, the adaptive resistance (analogues to the acquired immunity in multicellular organisms) serves as a second line of defence, which is very efficient and phage-specific.
Interestingly, it was observed that the bacterial anti-phage mechanisms are generally present in a genomic array, known as ‘defence islands’ [20]. The ‘defence islands’ are enriched in putative operons and contain numerous overrepresented genes encoding diverged variants of antiviral defence systems. Moreover, scientific evidence and characteristic operonic organisation of ‘defence islands’ show that many more anti-phage mechanisms are yet to be discovered [21, 22, 23, 24].
Although bacteria have developed several resistance mechanisms against phages, phages can circumvent bacterial anti-phage mechanisms on the grounds of their genomic plasticity and rapid replication rates. These counterstrategies include point mutations in specific genes and genome rearrangements that allow phages to evade bacterial antiviral systems such as CRISPR/Cas arrays by using anti-CRISPR proteins and abortive infection by hijacking bacterial antitoxins, as well as escaping from adsorption inhibition and restriction-modification mechanisms [15, 16, 17, 18].
This chapter will comment on the genetic basis of bacterial resistance to phages and different strategies used by phages to evade bacterial resistance mechanisms.
3. Preventing phage adsorption and phage’s counterstrategy
Phage adsorption to host-specific receptors on the cell surface is the initial step of the infection and host-phage interaction. Depending on the nature of bacteria, whether it is Gram-positive or Gram-negative proteins, lipopolysaccharides, teichoic acids and other cell surface structures can serve as irreversible phage-binding receptors [19]. These receptors might be present in the cell wall, bacterial capsules, slime layers, pili or flagella [25].
Bacteria have acquired various barriers to inhibit phage adsorption, such as blocking of phage receptors, production of extracellular matrix (e.g. capsule, slime layers) and production of competitive inhibitors [26, 27, 28, 29, 30, 31]. The diversity of phage receptors in the host is influenced by co-evolutionary adaptations of phages to overcome these barriers [32]. This includes diversity-generating retroelements (DGRs) and phase variation mechanisms causing phenotypical differences within the bacterial colony [7, 33, 34].
Phase variation is a heritable, yet reversible process regulating gene expression in bacteria; genes can switch between a functional (expression) and a non-functional state leading to phenotypical variations within the bacterial population even when strains have identical genotype. Sørensen et al. [35] investigated the underlying resistance mechanism of Campylobacter jejuni (NCTC11168) to phage F336. They have discovered that phage F336 relies on the hypervariable O-methyl phosphoramidate (MeOPN) modification of capsular polysaccharides (CPS) for successful adsorption to the bacterial surface. Nevertheless, loss of MeOPN receptor on the bacterial cell surface due to phase variation in the cj1421 gene encoding the MeOPN-GalfNAc transferase (MeOPN transferase attaches MeOPN to GalfNAc and Hep side chains of CPS) results in phage resistance [35, 36].
DGRs are genetic elements diversifying DNA sequences and the proteins they encode ultimately mediating the evolution of ligand-receptor interactions. Error-prone DGRs and random mutations in the bacterial genes encoding cell surface receptors lead to the alternation and change in the structural composition of the phage receptors, making them non-complementary to the phage’s anti-receptors, known as receptor-binding proteins (RBP) [34] (Figure 1(1)).
Figure 1.
Bacterial defence mechanisms preventing phage adsorption and phage’s counteradaptations. (1) Phage adsorption to a host-specific receptor site on a host cell surface. Bacterium evolves phage resistance by the modification of these cell surface receptors; phage is incapable of binding to the altered receptor. (2) Phage’s adaptation to these modifications through mutations in receptor-binding protein gene that leads to the co-evolution of bacterial genetic variation. Bacteria are also capable of producing proteins that mask the phage recognition site receptors (3 and 4), thus making the receptor inaccessible for phage adsorption [28, 29, 30, 31]. Image courtesy of springer nature: https://www.ncbi.nlm.nih.gov/pubmed/20348932.
Yet, phage’s replication is exceedingly error-prone, therefore causing many random mutations in the genes encoding the RBP or tail fibres. Phages also possess DGRs that mediate phage’s tropism by accelerating the variability in the receptor-coding genes through reverse transcription process [37]. The changes in the nucleotide sequence in the RBP-coding gene may ultimately lead to the adaptation to the modified receptor (Figure 1(2)), thus the ability to adsorb and infect the bacterial cell.
Unsurprisingly, bacteria also exhibit different strategies to block their receptors [28, 29, 30, 31].
Figure 1(4) demonstrates the findings from studies conducted on Staphylococcus aureus by Nordstrom and Forsgren [38]. Mutants of Staphylococcus aureus producing higher anticomplementary protein A were found to adsorb fewer phages than Staphylococcus aureus mutants with scarce of protein A, which had an apparent increased ability to adsorb phages [38]. These findings indicate that some bacteria, including Staphylococcus aureus, are capable of production of surface proteins that mask the phage receptors making them inaccessible for phage recognition and attachment (Figure 1(3)).
Receptors located on bacterial cell surface serve a vital role in bacterial metabolism; they may function as membrane porins, adhesions or chemical receptors [19]. Therefore, mutation or complete loss of the receptor might be lethal for bacteria. To inhibit phage adsorption, bacteria can produce surface molecules, such as exopolysaccharides.
Exopolysaccharides are extracellular polysaccharides acting as a physical barrier, composing slime or capsules surrounding bacterial cells that lead to inaccessible host receptors for efficient phage adsorption [39] (Figure 2). Studies conducted by Looijesteijn et al. [40] shown that exopolysaccharides produced by Lactococcus lactis function as external protection from phages and the cell wall destructing lysozyme, due to masked cell surface receptors [40].
Figure 2.
Bacterial strategies to inhibit phage adsorption and phage strategies to access host receptors. Some bacteria are capable of the production of exopolysaccharides, which act as an outer shield, protecting a cell from the phage infection [28, 29, 30, 31]. If the phage does not possess any polysaccharide-degrading enzymes, it cannot access the host cell membrane receptor. However, some phages evolved mechanisms allowing them to recognise these extracellular matrixes and degrade them by the means of hydrolases and lyases [15, 16, 17, 18]. Image courtesy of Springer Nature: https://www.ncbi.nlm.nih.gov/pubmed/20348932.
Nevertheless, some phages evolved mechanisms allowing them to recognise these extracellular matrixes and degrade them by utilising hydrolases and lyases (Figure 2) [15, 16, 17, 18]. The polysaccharide-degrading enzymes allow phages to gain access to the receptor that may lead to the viral propagation. They are commonly present bound to the RBPs or exist as free soluble enzymes from previously lysed bacterial cells [41].
4. Preventing phage DNA entry and phage’s counteradaptations
If phage bypasses primary antiviral strategies, it is now able to initiate infection by adsorption to a specific receptor site on a host cell surface through phage RBP [42, 43]. Upon interaction with the cell receptors, the phage injects its genetic material (single or double-stranded DNA or RNA) into the cytoplasm of the host. Depending on the nature of the phage and growth conditions of the host cell, it follows one of the two life cycles: lytic or lysogenic (Figure 3).
Figure 3.
Lytic and lysogenic life cycles of a temperate coliphage λ that infects Escherichia coli [44, 45]. cos—cohesive sites: the joining ends that circularise the linear phage λ DNA. Image courtesy of Springer Nature: https://www.nature.com/articles/nrg1089.
In the lytic cycle, virulent phages degrade host’s genome leading to the biosynthesis of viral proteins and nucleic acids for the assembly of phage progeny. Eventually, the bacterial cell lysis, releasing a multitude of newly assembled phages, is ready to infect a new host cell [46].
In contrast, temperate phages might enter the lytic or lysogenic cycle, if the host cell exists in adverse environmental conditions that could potentially limit the number of produced progeny (Figure 3 demonstrates typical lifecycle of temperate phage using coliphage λ as an example) [44, 45]. In the lysogenic phase, repressed phage genome integrates into the bacterial chromosome as a prophage. This process causes the proliferation of prophage during replication and binary fission of bacterial DNA.
Prophage only expresses a repressor protein-coding gene. The repressor protein binds to the operator sites of the other genes and ultimately inhibits synthesis of phage enzymes and proteins required for the lytic cycle.
When the synthesis of the repressor protein stops or if it becomes inactivated, a prophage may excise from the bacterial chromosome, initiating a lytic cycle (induction) which leads to the multiplication and release of virulent phages and lysis of a host cell [44, 45].
If the phage remains in the nearly dormant state (prophage), the lysogenic bacterium is immune to subsequent infection by other phages that are the same or closely analogous to the integrated prophage by means of Superinfection exclusion (Sie) systems [47].
Sie systems are membrane-associated proteins, generally, phage or prophage encoded, that prevent phage genome entry into a host cell [47]. Figure 4 shows the role of Sie system (proteins Imm and Sp) in blocking phage T4 DNA entry into Gram-negative Escherichia coli. Despite successful attachment to the phage-specific receptor, phage DNA is directly blocked by Imm protein from translocating into the cytoplasm of the cell. Sp system, on the other hand, prevents the degradation of the peptidoglycan layer by inhibiting the activity of T4 lysozyme [26, 27, 28, 29, 30, 31, 48].
Figure 4.
Superinfection exclusion systems preventing phage DNA entry in Gram-negative Escherichia coli. (a). Standard T4 phage: upon attachment to phage-receptor on the surface of the host cell, an inner-membrane protein aids the translocation of phage DNA into the cell’s cytoplasm. (b) Imm encoding phage T4: Imm protein directly blocks the translocation of the phage DNA into the cytoplasm of the cell. (c) Imm and Sp encoding phage T4: phage DNA is prevented from entering the cell’s cytoplasm by Imm; and Sp protein prevents degradation of the peptidoglycan layer by inhibiting the activity of T4 lysozyme [28, 29, 30, 31]. Image courtesy of Springer Nature: https://www.ncbi.nlm.nih.gov/pubmed/20348932.
5. Host strategies to cleave invading genomes and evolutionary tactics employed by phages to bypass these antiviral mechanisms
The evolution of bacterial genomes allowed bacteria to acquire vast mechanisms interfering with every step of phage infection. In a case where a phage succeeded to inject its viral nucleic acid into a host cell, bacteria possess a variety of nucleic acid degrading systems such as restriction-modification (R-M) systems and CRISPR/Cas that protect bacteria from the phage invasion.
5.1 Restriction-modification systems
It has been reported that R-M systems can significantly contribute to bacterial resistance to phages [49].
R-M systems incorporate activities of methyltransferases (MTases) that catalyse the transfer of a methyl group to DNA to protect self-genome from a restriction endonuclease (REase) cleavage and REases, which recognise and cut foreign unmethylated double-stranded DNA at specific recognition sites, commonly palindromic. To protect self-DNA from the degradation, methylases tag sequences recognised by the endonucleases with the methyl groups, whereas unmethylated phage (nonself) DNA is cleaved and degraded (Figure 5) [26, 27, 50, 51, 52].
Figure 5.
General representation of the bacterial restriction-modification (R-M) systems providing a defence against invading phage genomes. R-M systems consist of two contrasting enzymatic activities: a restriction endonuclease (REase) and a methyltransferase. REase recognises and cuts nonself unmethylated double-stranded DNA at specific recognition sites, whereas MTase adds methyl groups to the same genomic recognition sites on the bacterial DNA to protect self-genome from REase cleavage [50, 51]. Image courtesy of: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591985/.
R-M systems are diverse and ubiquitous among bacteria. There are four known types of R-M within bacterial genomes (Figure 6). Their classification is mainly based on R-M system subunit composition, sequence recognition, cleavage position, cofactor requirements and substrate specificity [26, 27, 50, 51].
Figure 6.
Four distinct types of restriction-modification (R-M) systems. (a) Type I R-M system is composed of three subunits forming a complex: hsdR (restriction), hsdM (modification) and hsdS (specificity subunit that binds to an asymmetrical DNA sequence and determines the specificity of restriction and methylation). Two hsdM subunits and one hsdS subunit are involved in methylation of self-DNA. On the other hand, two complexes of hsdR, hsdM and hsdS (where each complex consists of two hsdR, two hsdM and one hsdS subunit) bind to the unmethylated recognition sites on phage DNA and cleave the DNA at random, far from their recognition sequences. Both reactions—methylation and cleavage—require ATP. (b) Type II R-M system is composed of two distinct enzymes: palindromic sequence methylating methyltransferase (mod) and endonuclease (res) that cleave unmethylated palindromic sequences close to or within the recognition sequence. (c) Type III R-M system is formed of methyltransferase (mod) and endonuclease (res) that form a complex. Methyltransferase transfers methyl group to one strand on the DNA, whereas two methyltransferases (endonuclease complexes) act together to bind to the complementary unmethylated recognition sites to cleave the DNA 24–26 bp away from the recognition site. (d) Type IV R-M system contains only endonuclease (res) that recognises methylated or modified DNA. Cleavage occurs within or away from the recognition sequences [26, 27, 50, 51]. Image courtesy of: https://www.annualreviews.org/doi/abs/10.1146/annurev-virology-031413-085500?journalCode=virology.
Due to the diversity of R-M systems, phages acquired several active and passive strategies to bypass cleavage by REases. Passive mechanisms include reduction in restriction sites, modification and change of the orientation of restriction sites, whereas more specific, active mechanisms include masking of restriction sites, stimulation of MTase activity on phage genome or degradation of an R-M system cofactor (Figure 7) [15, 16, 17, 18].
Figure 7.
Phage’s passive and active strategies to bypass restriction-modification (R-M) systems. (a) Phages that possess fewer restriction sites in their genome are less prone to DNA cleavage by the host restriction endonuclease (REase). (b) Occasionally phage DNA might be modified by bacterial methyltransferase (MTase) upon successful injection into a host cell. Methylated recognition sites on viral DNA are, therefore, being protected from the cleavage and degradation by REase, leading to the initiation of the phage’s lytic cycle. In addition, some phages encode their own MTase that is cooperative with the host REase; thus viral DNA cannot be recognised as nonself. (c) Some phages, for example, coliphage P1, while injecting its DNA into a host cell, it also co-injects host-genome-binding proteins (DarA and DarB) that mask R-M recognition sites. (d) Phages such as Coliphage T7 possess proteins that can mimic the DNA backbone. Ocr, a protein expressed by Coliphage T7, mimics the DNA phosphate backbone and has a high affinity for the EcoKI REase component, thereby interfering with R-M system. (e) In addition, some phages (e.g. Ral protein of Coliphage λ) can also stimulate activity of the bacterial modification enzyme in order to protect own DNA from the recognition by the bacterial REase as nonself. The peptide Stp encoded by Coliphage T4 can as well disrupt the structural conformation of the REase-MTase complex [15, 16, 17, 18]. Image courtesy of: https://www.nature.com/articles/nrmicro3096.
Fewer restriction sites in the evading genome lead to the selective advantage of this phage as its DNA is less prone to cleavage and degradation by the host REase (Figure 7a). Also, some phages incorporate modified bases in their genomes that may lead to successful infection of the host cell as REase may not recognise the new sequences in the restriction sites. A decrease in the effective number of palindromic sites in DNA or change in the orientation of restriction-recognition sites can affect R-M targeting. Alternatively, the recognition sites within the viral genome can be too distant from each other to be recognised and cleaved by the REase [15, 16, 17, 18, 53].
Interestingly, phage genome might be methylated by bacterial MTase upon successful injection into a host cell. Methylated recognition sites on viral genomes are therefore being protected from the cleavage and degradation by REase, leading to the initiation of the phage’s lytic cycle. Viral progeny remains insensitive to this specific bacterial REase until it infects a bacterium that possesses a different type of REase, in which case the new progeny will become unmethylated again and will, therefore, be sensitive to the R-M system of the cognate bacterium [28, 29, 30, 31].
The fate of the host cell chiefly confides in the levels of R-M gene expression and ultimate proportion of the R-M enzymes and their competition for the sites in the invading phage genome [52].
Furthermore, some phages encode their own MTase that is cooperative with the host REase, and thereby viral DNA cannot be recognised as nonself. Phages can also stimulate the activity of host modification enzymes that can rapidly methylate viral DNA, thus protecting it from the activity of REase.
Alternatively, phages can bypass R-M systems by masking restriction sites. For example (Figure 7c), coliphage P1, while injecting its DNA into a host cell, it also co-injects host-genome-binding proteins (DarA and DarB) that mask R-M recognition sites [53, 54].
As shown on an example of a Coliphage T7 (Figure 7d), some phages code for proteins that directly inhibit REase. Coliphage T7 possesses proteins that can mimic the DNA backbone. Ocr, a protein expressed by Coliphage T7, directly blocks the active site of some REases by mimicking 24 bp of bent B-form DNA, and it has a high affinity for the EcoKI REase component, thereby interfering with R-M system [53].
Lastly, phage-bacteria arm race allowed phages to gain capabilities of degrading necessary cofactors of R-M systems. For instance, coliphage T3 encodes S-adenosyl-l-methionine hydrolase that destroys an essential host R-M cofactor (the S-adenosyl-l-methionine). The removal of this necessary co-factor will lead to the inhibition of the REase, thereby successfully infecting the host cell [15, 16, 17, 18].
5.2 CRISPR/Cas system
CRISPR along with CRISPR-associated (Cas) proteins is the type of adaptive heritable ‘immunity’ of bacteria, thus very specific and effective; and it is prevalent within the bacterial domain [55]. The CRISPR are DNA loci consisting of short palindromic repeats (identical in length and sequence), interspaced by segments of DNA sequences (spacer DNA) derived from previous exposures to phages. The spacer DNA sequences act as a ‘memory’, allowing bacteria to recognise and destroy specific phages in a subsequent infection. Genes encoding Cas proteins are adjacent to CRISPR loci [56].
Although some studies have suggested that CRISPRs can be used for pathogen subtyping [57], it has been found that CRISPR typing is not useful for the epidemiological surveillance and outbreak investigation of Salmonella typhimurium [58].
The CRISPR/Cas phage resistance is mediated in three-step stages: adaptation (acquisition), where spacer phage-derived DNA sequences are incorporated into the CRISPR/Cas system; expression, where cas gene expression and CRISPR transcription lead to pre-CRISPR RNA (pre-crRNA) that is then processed into CRISPR RNA (crRNA); and interference, during which the crRNA guides Cas proteins to the target (subsequently invading DNA) for the degradation. The cleavage of the target (proto-spacer) depends on the recognition of complementary sequences in spacer and protospacer [59, 60].
CRISPR/Cas systems have been classified into three major types: Types I, II and III, which are further divided into subtypes that require different types of Cas proteins. Although the CRISPR/Cas array is diverse among the bacteria and it is continuously co-evolving in response to the host-phage interactions, the defence activity in all three types of the CRISPR is comparable [21, 22, 23] Figure 8 illustrates the defence mechanisms in three distinct CRISPR/Cas arrays.
Figure 8.
Image showing mechanisms of adaptation, expression and interference in three different types of CRISPR/Cas arrays. Type I and Type II CRISPR/Cas arrays rely on the protospacer adjacent motif (PAM), contained within phage nucleic acid, to ‘select’ the phage-derived protospacer. Next steps in the adaptation stage are similar in all three types; protospacer is incorporated by Cas 1 and Cas2 proteins into the bacterial genome at the leader end of the CRISPR loci to form a new spacer. In expression step, CRISPR loci are transcribed into pre-crRNA. The crRNA processing and interference stage is distinct in each type of the CRISPR/Cas system. In Type I, the multisubunit CRISPR-associated complex for antiviral defence (CASCADE) binds crRNA to locate the target, and with the presence of Cas3 protein, the invading target genome is degraded whereas in Type II, Cas9 protein is essential in the processing of the crRNA. TracrRNA recognises and attaches to the complementary sequences on the repeat region that is then cut by RNase III in the presence of Cas9. Lastly, in Type III, processing of pre-crRNA into crRNA is dependent upon the activity of Cas6. Mature crRNA associated with Csm/Cmr complex targets foreign DNA or RNA for the degradation [21, 22, 23]. Image courtesy of: https://www.nature.com/articles/nrmicro2577.
The Type II, CRISPR/Cas9, which was first identified in Streptococcus pyogenes, gained considerable interest within scientific studies as a precise genome editing tool. CRISPR/Cas9 system is unique; a single Cas 9 protein (in addition to prevalent Cas 1 and Cas 2) is involved in the processing of crRNA and destruction of the target viral DNA [56, 61].
In the adaptation stage, phage-derived protospacer (snippet of DNA from the invading phage) is incorporated into the bacterial genome at the leader end of the CRISPR loci. In expression phase, the Cas9 gene expresses Cas9 protein possessing DNA cleaving HNH and RuvC-like nuclease domains; CRISPR locus is then transcribed and processed into mature crRNA. Finally, in interference step, the complex consisting of Cas9, crRNA and separate trans-activating crRNA (tracrRNA) cleave 20 base pairs crRNA-complementary target sequence that is adjacent to the protospacer adjacent motif (PAM) [62].
To bypass CRISPR/Cas that has an incredibly dynamic rate of evolution, phages acquired array of strategies to succeed in propagation; this includes mutations in the protospacers or in the PAM sequences and expression of anti-CRISPR proteins, and even some phages encode their own functional CRISPR/Cas systems [15, 16, 17, 18, 63].
Phages can evade interference step of Type I and Type II CRISPR/Cas system by a single point mutation or deletion in their protospacer region or in the PAM sequence (Figure 9). Phages with single-nucleotide substitutions or deletions positioned close to PAM sequence can bypass the CRISPR/Cas activity and complete their lytic cycles; in contrast, phages with multiple mutations at PAM-distal protospacer positions do not [15, 16, 17, 18, 28, 29, 30, 31].
Figure 9.
Evasion by mutation. Mutations in the phage protospacers or in the PAM sequences allow the phage to escape interference step of the CRISPR/Cas system that would lead to the degradation of the phage genome [15, 16, 17, 18]. Adapted image courtesy of: https://www.nature.com/articles/nrmicro3096.
In some circumstances, however, although the phage successfully evades CRISPR/Cas interference, the host cell may survive by the acquisition of new spacer sequences (derived from invading phage) into their own CRISPR/Cas system. This new spacer provides the bacterium with an accelerated spectrum of phage resistance [15, 16, 17, 18].
Prophages integrated within Pseudomonas aeruginosa possess genes that encode anti-CRISPR proteins directly suppressing CRISPR/Cas-mediated degradation of the phage genome (Figure 10). According to Wiedenheft [64], these proteins might interrupt CRISPR RNA processing by preventing mature crRNA from binging to the crRNA-guide complex or by preventing the assembled crRNA-guided complex from interacting with target substrates through binding to it [64].
Figure 10.
Anti-CRISPR proteins expressed against CRISPR subtype I-F systems. Temperate phages such as Pseudomonas aeruginosa possess genes encoding anti-CRISPR proteins that directly interfere with the bacterial CRISPR/Cas system [15, 16, 17, 18]. Adapted image courtesy of: https://www.nature.com/articles/nrmicro3096.
Prophages do not only contribute to bacterial resistance to invading phages, they can also encode proteins that contribute to bacterial virulence and antimicrobial resistance [58, 66].
Bacteria can also resist phages by possessing phage-inducible chromosomal islands (PICI) which prevent phage replication. Nevertheless, phages evolved their genomes to overcome this very specific antiviral strategy. For example, Vibrio cholerae ICP1 phages possess their own CRISPR/Cas systems that inactivate PICI-like elements (PLE) in Vibrio cholerae (Figure 11). Studies conducted by Naser et al. [67] have shown that phage CRISPR arrays have evolved by the acquisition of new spacers targeting diverse regions of PLEs carried by Vibrio cholerae strains. Furthermore, the addition of the new spacers within phage CRISPR/Cas loci enables the phages to expand their ability to counter PLE-mediated phage defence of diverse Vibrio cholerae strains [67].
Figure 11.
Phage-encoded CRISPR/Cas systems in Vibrio cholerae ICP1 phages. Upon adsorption and injection of viral genome into a host cell, phage crRNAs and CRISPR/Cas complexes are expressed and target phage-inducible chromosomal island (PICI) in the host genome; in the Vibrio cholerae, they are termed as PICI-like elements (PLE). If the spacers within phage CRISPR locus are complementary to the bacterial PLE, the CRISPR machinery is then able to specifically target this genetic element and inactivate it, leading to the viral propagation. However, in the absence of such targeting, phage CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the PLE to restore phage replication [15, 16, 17, 18, 65]. Adapted image courtesy of: https://www.nature.com/articles/nrmicro3096.
Abortive infection (Abi) systems promote cell death of the phage-infected bacteria, inhibiting phage replication and providing protection for bacterial populations [68].
Abi systems require both toxins and antagonistic antitoxins. Antitoxins are proteins or RNAs that protect bacterial cell from the activity of toxins in a typical cell life cycle, whereas toxins are the proteins encoded in toxin-antitoxin locus that disrupt cellular metabolism (translation, replication and cell wall formation), causing cell death. During an infection, the expression of the antitoxin encoding gene is suppressed, leading to the lethal activation of the toxin [69]. Figure 12 illustrates the mechanism of Abi systems in Escherichia coli [70].
Figure 12.
Abortive infection (Abi) systems in Escherichia coli. The Rex system is a two-component Abi system. A phage protein-DNA complex (formed during phage replication) activates the sensor protein RexA, which in turn activates RexB. RexB is an ion channel that causes depolarisation of the bacterial membrane leading to cell death [28, 29, 30, 31]. Image courtesy of Springer Nature: https://www.ncbi.nlm.nih.gov/pubmed/20348932.
Interestingly, phages evolved an array of tactics to circumvent Abi systems. This includes mutations in specific phage genes and encoding own antitoxin molecules that suppresses bacterial toxin [15, 16, 17, 18]. Figure 13 provides a broad overview of the strategies employed by the phages to by-pass Abi systems.
Figure 13.
Escaping abortive infection mechanisms. (a) In a typical cell life cycle, antitoxins protect bacterial cell from the activity of toxins. (b) During phage infection, the expression of antitoxin encoding gene is suppressed, leading to the lethal activation of the toxin. (c) Mutations in certain phage genes can lead to escaping Abi systems activity, thereby a successful viral propagation without killing the host cell. (d) Some phages encode molecules that functionally replace the bacterial antitoxins, thus suppressing toxin activity and avoiding host cell death [15, 16, 17, 18]. Image courtesy of: https://www.nature.com/articles/nrmicro3096.
Bacteria-phage interaction is therefore very complex, and it is crucial to understand the molecular basis of this interaction and how bacteria and phages ‘fight’ each other. It has been reported that Anderson Phage Typing System of Salmonella Typhimurium can provide a valuable model system for study of phage-host interaction [71].
7. The potential application of phages as antibacterial therapeutics
The rapid emergence and dissemination of MDR bacteria seriously threaten global public health, as, without effective antibiotics, prevention and treatment of both community- and hospital-acquired infections may become unsuccessful and lead to widespread outbreaks.
Carbapenems and colistin are antibiotics of last resort, generally reserved to treat bacteria which are resistant to all other antibiotics. Until not long ago, colistin resistance was only described as chromosomal, however, in 2016 Liu et al. reported the emergence of the first plasmid-mediated colistin resistance mechanism, MCR-1, in Enterobacteriaceae [72]. Furthermore, the increasing occurrence of colistin resistance among carbapenem-resistant Enterobacteriaceae has also been reported [73]. This is of significant concern as infections caused by colistin and carbapenem-resistant bacteria are very challenging to treat and control, as the treatment options are greatly limited or non-existent. Thus, the discovery and development of alternative antimicrobial therapeutics are the highest priorities of modern medicine and biotechnology.
Phages should be considered as great potential tools in MDR pathogens as they are species-specific (specificity prevents damage of normal microbiota), thus harmless to human; they have fast replication rate at the site of infection, and their short genomes can allow to further understand various molecular mechanisms implied to ‘fight’ bacteria. In addition, this understanding can enable scientists to ‘manipulate’ viral genomes and engineer a synthetic phage that combines the antibacterial characteristics of multiple phages into a single genome.
The escalating need for new antimicrobial agents attracted new attention in modern medicine, proposing several potential applications of phages as antibacterial therapeutics including phage therapy, phage lysins and genetically-engineered phages.
7.1 Phage therapy
Phage therapy utilises strictly lytic phages that have bactericidal effect. As phages are host-specific, ‘phage cocktails’ containing multiple phages can broaden range of target cells. Nevertheless, selection of suitable phages is at the paramount to the successful elimination of clinically important pathogens, and it includes avoidance of adverse effects, such as anaphylaxis (adverse immune reaction) [74].
7.2 Phage-derived enzymes: lysins
In order to hydrolyse and degrade the bacterial cell wall, phages possess lysins.
The spectrum of efficiency of natural lysins (derived from naturally occurring phages) is generally limited to Gram-positive bacteria; however, recombinant lysins have shown an ability to destabilise the outer membrane of Gram-negative bacteria and ultimately lead to rapid death of the target bacteria [74].
7.3 Bioengineered phages
Bioengineered phages have the potential to solve inherent limitations of natural phages such as narrow host range and evolution of resistance. Various genetic engineering methods have been proposed to design phages with extended antimicrobial properties such as homologous recombination, phage recombineering of electroporated DNA, yeast-based platform, Gibson assembly and CRISPR/Cas genome editing [75].
Engineering of synthetic phages could be tailored to enhance the antibiotic activity, to reverse antibiotic resistance or to create sequence-specific antimicrobials [74].
8. Conclusions
The antagonistic host-phage relationship has led to the evolution of exceptionally disperse phage-resistance mechanisms in the bacterial domain, including inhibition of phage adsorption, prevention of nucleic acid entry, Superinfection exclusion, cutting phage nucleic acids via restriction-modification systems and CRISPR, as well as abortive infection.
Evolvement of these mechanisms has been induced by constant parallel co-evolution of phages as they attempt to coexist. To survive, phages acquired diverse counterstrategies to circumvent bacterial anti-phage mechanisms such as adaptations to new receptors, digging for receptors and masking and modification of restriction sites and point mutations in specific genes and genome rearrangements that allow phages to evade bacterial antiviral systems such as CRISPR/Cas arrays, as well as mutations in specific genes to bypass abortive infection system. Conclusively, the co-evolving genetic variations and counteradaptations, in both bacteria and phages, drive the evolutionary bacteria-host arm race.
Besides, accumulating evidence shows that phages contribute to the antimicrobial resistance through horizontal gene transfer mechanisms. Indeed, many bacterial strains have become insensitive to the conventional antibiotics, posing a growing threat to human; and although in the past, western counties withdrew phage therapy in response to the discovery of therapeutic antibiotics, now, phage therapy regains an interest within the research community. There are apparent advantages of phage therapy, such as specificity, meaning only target bacteria would encounter lysis, but not healthy microbiota inhabiting human’s system. Additionally, ‘phage cocktails’, containing multiple bacteria-specific phages, could overcome the issue of phage-resistance as phages do adapt to these resistance mechanisms. However, ‘phage cocktails’ would require large numbers of phages that would have to be grown inside pathogenic bacteria in the laboratory, putting laboratory staff and the environment at risk.
Alternatively, building up the understanding of host-phage interactions and ‘the war between bacteria and phages’ could potentially lead to defeating antimicrobial resistance by designing synthetic phages that can overcome the limitations of phage therapy.
Acknowledgments
Dr Manal Mohammed is funded by a Quinton Hogg start-up award, University of Westminster.
Abbreviations
Abi
abortive infection
CPS
capsular polysaccharides
CRISPR
clustered regularly interspaced short palindromic repeats
crRNA
crispr RNA
DGR
diversity-generating retroelement
DNA
deoxyribonucleic acid
MDR
multidrug-resistant
MeOPN
O-methyl phosphoramidate
MTase
methyltransferase
PAM
protospacer adjacent motif
PICI
phage-inducible chromosomal island
PLE
PICI-like element
RBP
receptor-binding protein
REase
restriction endonuclease
R-M
restriction-modification
RNA
ribonucleic acid
Sie
superinfection exclusion
tracrRNA
trans-activating crRNA
\n',keywords:"bacteria-phage arms race, CRISPR system, anti-CRISPR system, superinfection exclusion (Sie), restriction-modification, abortive infection (Abi)",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/69255.pdf",chapterXML:"https://mts.intechopen.com/source/xml/69255.xml",downloadPdfUrl:"/chapter/pdf-download/69255",previewPdfUrl:"/chapter/pdf-preview/69255",totalDownloads:750,totalViews:0,totalCrossrefCites:1,dateSubmitted:"July 15th 2018",dateReviewed:"June 4th 2019",datePrePublished:"October 31st 2019",datePublished:null,dateFinished:null,readingETA:"0",abstract:"The rapid emergence and dissemination of multidrug-resistant (MDR) bacteria represents a worldwide crisis concerning that humankind is re-entering the ‘pre-antibiotics’ era. Before the discovery of antibiotics, bacteriophage therapy was widely enforced to combat bacterial infections. However, the discovery of penicillin in 1940 and other novel antibiotics replaced phage therapy, and they are being used as the first line of defence against pathogenic bacterial infections. Factors such as selective pressure resulted in bacteria becoming insensitive to one or multiple antibiotics, frequently leading to limited treatment options. This prompted a renewal of interest to the phage therapy that remains dubious due to its disadvantages such as host specificity and the development of bacterial resistance against phages. Evolution of bacterial genomes allowed bacteria to acquire vast mechanisms interfering with phage infection such as inhibition of phage adsorption, prevention of phage entry, superinfection exclusion, restriction-modification and abortive infection. Interestingly, phages have developed diverse counterstrategies to circumvent bacterial anti-phage mechanisms including digging for receptors, adapting to new receptors and masking and modifying restriction sites. Understanding the complex dynamics of bacteria-phage interaction is a preliminary step towards designing synthetic phages that can overcome limitations of phage therapy and potentially lead to defeating MDR bacteria.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/69255",risUrl:"/chapter/ris/69255",signatures:"Beata Orzechowska and Manal Mohammed",book:{id:"7240",title:"Growing and Handling of Bacterial Cultures",subtitle:null,fullTitle:"Growing and Handling of Bacterial Cultures",slug:"growing-and-handling-of-bacterial-cultures",publishedDate:"December 4th 2019",bookSignature:"Madhusmita Mishra",coverURL:"https://cdn.intechopen.com/books/images_new/7240.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",editors:[{id:"204267",title:"Dr.",name:"Madhusmita",middleName:null,surname:"Mishra",slug:"madhusmita-mishra",fullName:"Madhusmita Mishra"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. The evolutionary phage-host arms race",level:"1"},{id:"sec_3",title:"3. Preventing phage adsorption and phage’s counterstrategy",level:"1"},{id:"sec_4",title:"4. Preventing phage DNA entry and phage’s counteradaptations",level:"1"},{id:"sec_5",title:"5. Host strategies to cleave invading genomes and evolutionary tactics employed by phages to bypass these antiviral mechanisms",level:"1"},{id:"sec_5_2",title:"5.1 Restriction-modification systems",level:"2"},{id:"sec_6_2",title:"5.2 CRISPR/Cas system",level:"2"},{id:"sec_8",title:"6. Overcoming host abortive infection systems: toxin-antitoxin",level:"1"},{id:"sec_9",title:"7. The potential application of phages as antibacterial therapeutics",level:"1"},{id:"sec_9_2",title:"7.1 Phage therapy",level:"2"},{id:"sec_10_2",title:"7.2 Phage-derived enzymes: lysins",level:"2"},{id:"sec_11_2",title:"7.3 Bioengineered phages",level:"2"},{id:"sec_13",title:"8. 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Available from: https://ecdc.europa.eu/en/publications-data/expert-consensus-protocol-colistin-resistance-detection-and-characterisation [Accessed: 13 May 2019]'},{id:"B74",body:'Kakasis A, Panitsa G. Bacteriophage therapy as an alternative treatment for human infections. A comprehensive review. International Journal of Antimicrobial Agents. 2019;53(1):16-21'},{id:"B75",body:'Monteiro R et al. Phage therapy: Going temperate? Trends in Microbiology. 2019;27(4):368-378. DOI: 10.1016/j.tim.2018.10.008'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Beata Orzechowska",address:null,affiliation:'
School of Life Sciences, College of Liberal Arts and Sciences University of Westminster, London, UK
School of Life Sciences, College of Liberal Arts and Sciences University of Westminster, London, UK
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The company was founded in Vienna in 2004 by Alex Lazinica and Vedran Kordic, two PhD students researching robotics. While completing our PhDs, we found it difficult to access the research we needed. So, we decided to create a new Open Access publisher. A better one, where researchers like us could find the information they needed easily. The result is IntechOpen, an Open Access publisher that puts the academic needs of the researchers before the business interests of publishers.
",metaTitle:"Our story",metaDescription:"The company was founded in Vienna in 2004 by Alex Lazinica and Vedran Kordic, two PhD students researching robotics. While completing our PhDs, we found it difficult to access the research we needed. So, we decided to create a new Open Access publisher. A better one, where researchers like us could find the information they needed easily. The result is IntechOpen, an Open Access publisher that puts the academic needs of the researchers before the business interests of publishers.",metaKeywords:null,canonicalURL:"/page/our-story",contentRaw:'[{"type":"htmlEditorComponent","content":"
We started by publishing journals and books from the fields of science we were most familiar with - AI, robotics, manufacturing and operations research. Through our growing network of institutions and authors, we soon expanded into related fields like environmental engineering, nanotechnology, computer science, renewable energy and electrical engineering, Today, we are the world’s largest Open Access publisher of scientific research, with over 4,200 books and 54,000 scientific works including peer-reviewed content from more than 116,000 scientists spanning 161 countries. Our authors range from globally-renowned Nobel Prize winners to up-and-coming researchers at the cutting edge of scientific discovery.
\\n\\n
In the same year that IntechOpen was founded, we launched what was at the time the first ever Open Access, peer-reviewed journal in its field: the International Journal of Advanced Robotic Systems (IJARS).
\\n\\n
The IntechOpen timeline
\\n\\n
2004
\\n\\n
\\n\\t
Intech Open is founded in Vienna, Austria, by Alex Lazinica and Vedran Kordic, two PhD students, and their first Open Access journals and books are published.
\\n\\t
Alex and Vedran launch the first Open Access, peer-reviewed robotics journal and IntechOpen’s flagship publication, the International Journal of Advanced Robotic Systems (IJARS).
\\n
\\n\\n
2005
\\n\\n
\\n\\t
IntechOpen publishes its first Open Access book: Cutting Edge Robotics.
\\n
\\n\\n
2006
\\n\\n
\\n\\t
IntechOpen publishes a special issue of IJARS, featuring contributions from NASA scientists regarding the Mars Exploration Rover missions.
\\n
\\n\\n
2008
\\n\\n
\\n\\t
Downloads milestone: 200,000 downloads reached
\\n
\\n\\n
2009
\\n\\n
\\n\\t
Publishing milestone: the first 100 Open Access STM books are published
\\n
\\n\\n
2010
\\n\\n
\\n\\t
Downloads milestone: one million downloads reached
\\n\\t
IntechOpen expands its book publishing into a new field: medicine.
\\n
\\n\\n
2011
\\n\\n
\\n\\t
Publishing milestone: More than five million downloads reached
\\n\\t
IntechOpen publishes 1996 Nobel Prize in Chemistry winner Harold W. Kroto’s “Strategies to Successfully Cross-Link Carbon Nanotubes”. Find it here.
\\n\\t
IntechOpen and TBI collaborate on a project to explore the changing needs of researchers and the evolving ways that they discover, publish and exchange information. The result is the survey “Author Attitudes Towards Open Access Publishing: A Market Research Program”.
\\n\\t
IntechOpen hosts SHOW - Share Open Access Worldwide; a series of lectures, debates, round-tables and events to bring people together in discussion of open source principles, intellectual property, content licensing innovations, remixed and shared culture and free knowledge.
\\n
\\n\\n
2012
\\n\\n
\\n\\t
Publishing milestone: 10 million downloads reached
\\n\\t
IntechOpen holds Interact2012, a free series of workshops held by figureheads of the scientific community including Professor Hiroshi Ishiguro, director of the Intelligent Robotics Laboratory, who took the audience through some of the most impressive human-robot interactions observed in his lab.
\\n
\\n\\n
2013
\\n\\n
\\n\\t
IntechOpen joins the Committee on Publication Ethics (COPE) as part of a commitment to guaranteeing the highest standards of publishing.
\\n
\\n\\n
2014
\\n\\n
\\n\\t
IntechOpen turns 10, with more than 30 million downloads to date.
\\n\\t
IntechOpen appoints its first Regional Representatives - members of the team situated around the world dedicated to increasing the visibility of our authors’ published work within their local scientific communities.
\\n
\\n\\n
2015
\\n\\n
\\n\\t
Downloads milestone: More than 70 million downloads reached, more than doubling since the previous year.
\\n\\t
Publishing milestone: IntechOpen publishes its 2,500th book and 40,000th Open Access chapter, reaching 20,000 citations in Thomson Reuters ISI Web of Science.
\\n\\t
40 IntechOpen authors are included in the top one per cent of the world’s most-cited researchers.
\\n\\t
Thomson Reuters’ ISI Web of Science Book Citation Index begins indexing IntechOpen’s books in its database.
\\n
\\n\\n
2016
\\n\\n
\\n\\t
IntechOpen is identified as a world leader in Simba Information’s Open Access Book Publishing 2016-2020 report and forecast. IntechOpen came in as the world’s largest Open Access book publisher by title count.
\\n
\\n\\n
2017
\\n\\n
\\n\\t
Downloads milestone: IntechOpen reaches more than 100 million downloads
\\n\\t
Publishing milestone: IntechOpen publishes its 3,000th Open Access book, making it the largest Open Access book collection in the world
We started by publishing journals and books from the fields of science we were most familiar with - AI, robotics, manufacturing and operations research. Through our growing network of institutions and authors, we soon expanded into related fields like environmental engineering, nanotechnology, computer science, renewable energy and electrical engineering, Today, we are the world’s largest Open Access publisher of scientific research, with over 4,200 books and 54,000 scientific works including peer-reviewed content from more than 116,000 scientists spanning 161 countries. Our authors range from globally-renowned Nobel Prize winners to up-and-coming researchers at the cutting edge of scientific discovery.
\n\n
In the same year that IntechOpen was founded, we launched what was at the time the first ever Open Access, peer-reviewed journal in its field: the International Journal of Advanced Robotic Systems (IJARS).
\n\n
The IntechOpen timeline
\n\n
2004
\n\n
\n\t
Intech Open is founded in Vienna, Austria, by Alex Lazinica and Vedran Kordic, two PhD students, and their first Open Access journals and books are published.
\n\t
Alex and Vedran launch the first Open Access, peer-reviewed robotics journal and IntechOpen’s flagship publication, the International Journal of Advanced Robotic Systems (IJARS).
\n
\n\n
2005
\n\n
\n\t
IntechOpen publishes its first Open Access book: Cutting Edge Robotics.
\n
\n\n
2006
\n\n
\n\t
IntechOpen publishes a special issue of IJARS, featuring contributions from NASA scientists regarding the Mars Exploration Rover missions.
\n
\n\n
2008
\n\n
\n\t
Downloads milestone: 200,000 downloads reached
\n
\n\n
2009
\n\n
\n\t
Publishing milestone: the first 100 Open Access STM books are published
\n
\n\n
2010
\n\n
\n\t
Downloads milestone: one million downloads reached
\n\t
IntechOpen expands its book publishing into a new field: medicine.
\n
\n\n
2011
\n\n
\n\t
Publishing milestone: More than five million downloads reached
\n\t
IntechOpen publishes 1996 Nobel Prize in Chemistry winner Harold W. Kroto’s “Strategies to Successfully Cross-Link Carbon Nanotubes”. Find it here.
\n\t
IntechOpen and TBI collaborate on a project to explore the changing needs of researchers and the evolving ways that they discover, publish and exchange information. The result is the survey “Author Attitudes Towards Open Access Publishing: A Market Research Program”.
\n\t
IntechOpen hosts SHOW - Share Open Access Worldwide; a series of lectures, debates, round-tables and events to bring people together in discussion of open source principles, intellectual property, content licensing innovations, remixed and shared culture and free knowledge.
\n
\n\n
2012
\n\n
\n\t
Publishing milestone: 10 million downloads reached
\n\t
IntechOpen holds Interact2012, a free series of workshops held by figureheads of the scientific community including Professor Hiroshi Ishiguro, director of the Intelligent Robotics Laboratory, who took the audience through some of the most impressive human-robot interactions observed in his lab.
\n
\n\n
2013
\n\n
\n\t
IntechOpen joins the Committee on Publication Ethics (COPE) as part of a commitment to guaranteeing the highest standards of publishing.
\n
\n\n
2014
\n\n
\n\t
IntechOpen turns 10, with more than 30 million downloads to date.
\n\t
IntechOpen appoints its first Regional Representatives - members of the team situated around the world dedicated to increasing the visibility of our authors’ published work within their local scientific communities.
\n
\n\n
2015
\n\n
\n\t
Downloads milestone: More than 70 million downloads reached, more than doubling since the previous year.
\n\t
Publishing milestone: IntechOpen publishes its 2,500th book and 40,000th Open Access chapter, reaching 20,000 citations in Thomson Reuters ISI Web of Science.
\n\t
40 IntechOpen authors are included in the top one per cent of the world’s most-cited researchers.
\n\t
Thomson Reuters’ ISI Web of Science Book Citation Index begins indexing IntechOpen’s books in its database.
\n
\n\n
2016
\n\n
\n\t
IntechOpen is identified as a world leader in Simba Information’s Open Access Book Publishing 2016-2020 report and forecast. IntechOpen came in as the world’s largest Open Access book publisher by title count.
\n
\n\n
2017
\n\n
\n\t
Downloads milestone: IntechOpen reaches more than 100 million downloads
\n\t
Publishing milestone: IntechOpen publishes its 3,000th Open Access book, making it the largest Open Access book collection in the world
\n
\n"}]},successStories:{items:[]},authorsAndEditors:{filterParams:{sort:"featured,name"},profiles:[{id:"6700",title:"Dr.",name:"Abbass A.",middleName:null,surname:"Hashim",slug:"abbass-a.-hashim",fullName:"Abbass A. Hashim",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/6700/images/1864_n.jpg",biography:"Currently I am carrying out research in several areas of interest, mainly covering work on chemical and bio-sensors, semiconductor thin film device fabrication and characterisation.\nAt the moment I have very strong interest in radiation environmental pollution and bacteriology treatment. The teams of researchers are working very hard to bring novel results in this field. I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. 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After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\r\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. He is an expert in structural, absorptive, catalytic and photocatalytic properties, in structural organization and dynamic features of ionic liquids, in magnetic interactions between paramagnetic centers. The author or co-author of 3 books, over 200 articles and reviews in scientific journals and books. He is an actual member of the International EPR/ESR Society, European Society on Quantum Solar Energy Conversion, Moscow House of Scientists, of the Board of Moscow Physical Society.",institutionString:null,institution:{name:"Semenov Institute of Chemical Physics",country:{name:"Russia"}}},{id:"62389",title:"PhD.",name:"Ali Demir",middleName:null,surname:"Sezer",slug:"ali-demir-sezer",fullName:"Ali Demir Sezer",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62389/images/3413_n.jpg",biography:"Dr. Ali Demir Sezer has a Ph.D. from Pharmaceutical Biotechnology at the Faculty of Pharmacy, University of Marmara (Turkey). He is the member of many Pharmaceutical Associations and acts as a reviewer of scientific journals and European projects under different research areas such as: drug delivery systems, nanotechnology and pharmaceutical biotechnology. Dr. Sezer is the author of many scientific publications in peer-reviewed journals and poster communications. Focus of his research activity is drug delivery, physico-chemical characterization and biological evaluation of biopolymers micro and nanoparticles as modified drug delivery system, and colloidal drug carriers (liposomes, nanoparticles etc.).",institutionString:null,institution:{name:"Marmara University",country:{name:"Turkey"}}},{id:"61051",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"100762",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"St David's Medical Center",country:{name:"United States of America"}}},{id:"107416",title:"Dr.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Texas Cardiac Arrhythmia",country:{name:"United States of America"}}},{id:"64434",title:"Dr.",name:"Angkoon",middleName:null,surname:"Phinyomark",slug:"angkoon-phinyomark",fullName:"Angkoon Phinyomark",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/64434/images/2619_n.jpg",biography:"My name is Angkoon Phinyomark. 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I had been a visiting research student at Faculty of Computer Science, University of Murcia, Murcia, Spain for three months.\n\nI have published over 40 papers during 5 years in refereed journals, books, and conference proceedings in the areas of electro-physiological signals processing and classification, notably EMG and EOG signals, fractal analysis, wavelet analysis, texture analysis, feature extraction and machine learning algorithms, and assistive and rehabilitative devices. I have several computer programming language certificates, i.e. Sun Certified Programmer for the Java 2 Platform 1.4 (SCJP), Microsoft Certified Professional Developer, Web Developer (MCPD), Microsoft Certified Technology Specialist, .NET Framework 2.0 Web (MCTS). 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