Fabrication of Magnetic-Antimicrobial-Fluorescent Multifunctional Hybrid Microspheres and Their Properties
2013

Ling-Han Xiao
College of Chemistry
MacDiarmid Laboratory
Jilin University
130021ChangchunChina

Tao Wang
College of Chemistry
MacDiarmid Laboratory
Jilin University
130021ChangchunChina

Tian-Yi Zhao email@example.com.-y.z.
College of Chemistry
MacDiarmid Laboratory
Jilin University
130021ChangchunChina

Xin Zheng email@example.com.
College of Chemistry
MacDiarmid Laboratory
Jilin University
130021ChangchunChina

Li-Ying Sun
College of Chemistry
MacDiarmid Laboratory
Jilin University
130021ChangchunChina

Ping Li
Feng-Qi Liu email@example.com.-q.l.
College of Chemistry
MacDiarmid Laboratory
Jilin University
130021ChangchunChina

Ge Gao
College of Chemistry
MacDiarmid Laboratory
Jilin University
130021ChangchunChina

Alideertu Dong
College of Chemistry
MacDiarmid Laboratory
Jilin University
130021ChangchunChina

College of Chemistry and Chemical Engineering
Inner Mongolia University
010021HohhotChina

Fabrication of Magnetic-Antimicrobial-Fluorescent Multifunctional Hybrid Microspheres and Their Properties

OPEN ACCESS Int. J. Mol. Sci
1414201310.3390/ijms14047391Received: 1 January 2013; in revised form: 26 February 2013 / Accepted: 7 March 2013 /International Journal of Molecular Sciences * Authors to whom correspondence should be addressed; E-Mails: email@example.com (G.G.); firstname.lastname@example.com (A.D.); 7392poly(glycidyl methacrylate)magneticantibacterialfluorescentmultifunctionalhybrid microspheres
Novel magnetic-antimicrobial-fluorescent multifunctional hybrid microspheres with well-defined nanostructure were synthesized by the aid of a poly(glycidyl methacrylate) (PGMA) template. The hybrid microspheres were fully characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD) and digital fluorescence microscope. The as-synthesized microspheres PGMA, amino-modified PGMA (NH 2 -PGMA) and magnetic PGMA (M-PGMA) have a spherical shape with a smooth surface and fine monodispersity. M-PGMA microspheres are super-paramagnetic, and their saturated magnetic field is 4.608 emu·g −1 , which made M-PGMA efficiently separable from aqueous solution by an external magnetic field. After poly(haxemethylene guanidine hydrochloride) (PHGH) functionalization, the resultant microspheres exhibit excellent antibacterial performance against both Gram-positive and Gram-negative bacteria. The fluorescence feature originating from the quantum dot CdTe endowed the hybrid microspheres with biological functions, such as targeted localization and biological monitoring functions. Combination of magnetism, antibiosis and fluorescence into one single hybrid microsphere opens up the possibility of the extensive study of multifunctional materials and widens the potential applications.

Introduction

Guanidinium antibacterial materials have recently attracted considerable scientific attention, due to their unique advantages, such as high antibacterial activity, high stability, ease of storage, reproducibility, non-corrosiveness, non-toxicity and low cost. They have been widely utilized in various fields, e.g., medical devices, hospitals, water purification systems, food packaging, food preservation and sanitation [1][2][3][4][5][6][7][8]. As for a contact biocide, the contact area of guanidinium materials with microorganisms is very crucial to their antibacterial efficiency. Reduction in the size of the guanidinium antibacterial materials can increase their specific surface area and provide more effective antimicrobial functional groups and, thus, can improve the antimicrobial efficiency. Nano-materials have shown huge potentials and good prospects for their application in many fields, due to their outstanding properties, such as small size, large surface area and high reactivity [9]. Thus, increase of the specific surface area via preparing the guanidinium antibacterial materials with a nano-size could be an effective method to improve the antibacterial properties [10].

However, guanidinium antibacterial nano-materials still have some drawbacks, such as the cumbersome recovery process. The antibacterial process is usually carried out in an aqueous solution, and then residual antimicrobial nano-material should be isolated from a large amount of the aqueous solution by an expensive separation procedure. Introduction of a magnetic component into the antibacterial system is a necessary and effective way to reduce the recovery cost, due to its unique properties in the magnetic separation technology [11,12]. Magnetic separation is an important technique, which separates and recovers magnetic particles or particles susceptible to magnetic fields through an external magnetic field [13,14]. Micro/nano-particles and biological particles could be separated from the aqueous solution quickly and easily by utilizing this technology [15][16][17]. The greatest advantage of this technique is to isolate the maximum target substance in the shortest time. Since the mid-1970s, magnetic separation technology has been widely applied in the bioprocessing and biomedical fields, such as enzyme immobilization, cell arrangement, protein separation and drug delivery [18].

Fluorescent labeling is an important biotechnology, which plays a key role in biological imaging and biological monitoring. With the in-depth study of the fluorescent substance, the scientists found that the quantum dots have many advantages compared to conventional organic fluorescein for good stability, wide excitation wavelength, stable high quantum yield, highly narrow emission peaks and other aspects. As a fluorescent probe, doped quantum dot fluorescent microspheres can play a pivotal role in biological imaging and biological monitoring. The quantum dot CdTe were adsorbed on the polymer surface by electrostatic adsorption, so that the polymer has fluorescence, which has targeted localization and biological monitoring functions [19].

In this paper, poly(glycidyl methacrylate) (PGMA) was chosen as the cladding object, due to its high reactivity caused by superficial epoxy groups and good resistance to acid, heat, weather and solvent. Multifunctional hybrid microspheres with magnetic, antibacterial and fluorescent function were obtained using a PGMA template, as shown in Figure 1. The as-synthesized microspheres could be effectively recovered, due to their magnetic character, and they could be used in several areas, such as biological imaging and biological monitoring, because of their fluorescent property. For an antimicrobial test, Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) were used as model bacteria, and hybrid microspheres exhibited distinguishable antibacterial activity against all of them.

Results and Discussion

Description

PGMA microspheres capable of responding to magnetic fields were prepared by dispersion polymerization of GMA in the presence of iron oxides, as reported by Daniel Horák, and the conditions for synthesis and properties of the resulting magnetic carriers were studied systematically. Subsequently [20], micron-sized, monodisperse, superparamagnetic, luminescent composite poly(glycidyl methacrylate) (PGMA) microspheres with functional amino-groups were successfully synthesized by Chang et al. [21]. The composite microspheres were bright enough, easily observed using a conventional fluorescence microscope and were easily separated from solution by magnetic decantation using a permanent magnet. Although authors claimed that these new multifunctional composite microspheres were promising in a variety of bio-analytical assays involving luminescence detection and magnetic separation, no analytical applications were reported. Soon afterward, Jing et al. synthesized multifunctional PGMA microspheres with the capability of recognizing and binding cancer cell by the aid of the magnetic and fluorescent PGMA microspheres as support [22]. Hela cells could be captured with these PGMA microspheres from their suspension and easily moved in the direction of the magnetic field. On the basis of these studies mentioned above, magnetic-fluorescent bifunctional PGMA microspheres herein were further functionalized with antibacterial guanidinium to obtain magnetic-antimicrobial-fluorescent multifunctional microspheres. In this work, the combination of magnetism and fluorescence, with antibacterial performance in one single entity has been utilized for the first time to produce multifunctional nanocomposites.

The multifunctional M-PGMA/PHGH-CdTe microspheres were skillfully fabricated by the encapsulation of magnetic PGMA nanoparticles with antibacterial PHGH and the fluorescent CdTe attached to the PHGH shell. Figure 1 represents vividly the synthetic procedure of the multifunctional M-PGMA/PHGH-CdTe microspheres. Firstly, the PGMA support microspheres were modified by ethylene diamine to yield NH 2 -PGMA microspheres, and the magnetic M-PGMA microspheres are subsequently prepared by in situ precipitation. The PHGH coated magnetic PGMA nanoparticles were obtained by the aid of epoxy chloropropane (ECH) as mediator. At last, the fluorescent quantum dot CdTe were attached to the outer PHGH shell by electrostatic adsorption to obtain microspheres. Introducing magnetic PGMA nanoparticles as templates for preparation of PHGH shell enhances the materials' surface area and provides an effective means for the separation of the antibacterial agents through the application of a magnetic field. In addition, the quantum dot fluorescent microspheres can play a pivotal role in biological imaging and biological monitoring as a fluorescent probe. Similar strategies can also be utilized for the fabrication and separation of other multifunctional materials.

SEM Test of the PGMA, NH 2 -PGMA and M-PGMA Microspheres

SEM was carried out to investigate the morphology, structure, surface state and particle size of the as-synthesized microspheres. Figure 2A-C present the representative SEM images of the prepared PGMA, NH 2 -PGMA and M-PGMA microspheres. PGMA, NH 2 -PGMA and M-PGMA microspheres are quasi-monodisperse, spherical, surficial smooth and solid, and the mean particle size is 1.64, 1.77 and 1.81 μm, respectively. The increase in particle size further implied the successful formation of each synthetic step. However, there is no significant difference in particle surface between NH 2 -PGMA and M-PGMA microspheres, suggesting that Fe 3 O 4 nanoparticles prepared by in situ deposition were mainly deposited inside of the microspheres.  Figure 3A,B presents the representative SEM images of the prepared M-PGMA/PHGH-CdTe microspheres. The microspheres are quasi-monodisperse, spherical, surficial smooth and solid, and the mean particle size is 2.0 μm. Figure 3C depicts TEM images of the M-PGMA/PHGH-CdTe microspheres. In Figure 3C, the typical core-shell structure of PHGH with CdTe coated magnetic PGMA microspheres can be discerned. The M-PGMA/PHGH-CdTe microspheres are obviously spherical in shape, and the modified iron oxide nanoparticles are in the center of the microspheres. It can be seen that the quantum dots CdTe, which the arrow annotations denote in Figure 3C, were almost spherical attached to the PHGH shell, as indicated in Figure 1. The compositional information of the EDX spectrum in Figure 3D exhibits the presence of C, N, O, Cl, Fe and Cd elements in the M-PGMA/PHGH-CdTe microspheres. The signal of the Fe element is readily detected from Figure 3D, providing powerful evidence that the Fe 3 O 4 has successfully deposited in PGMA microspheres. It is observed clearly for the tiny content of Cd coming from the quantum dot CdTe in the enlarged figure (inset, Figure 3D). The N and Cl peaks appearance confirm the formation of PHGH copolymer on the surface of the PGMA microspheres.   Figure 4A, the strong peaks at 1732 and 1256 cm −1 are ascribed to the carbonyl group characteristic absorption and the symmetric stretching vibrations of the epoxy group, respectively [23]. The peaks at 848 and 910 cm −1 are assigned to the asymmetric stretching vibration peak of the epoxy group. In Figure 4B, NH 2 -PGMA microspheres have shown the disappearance of the epoxy group-symmetric stretching vibration peaks at around 848 and 910 cm −1 compared with the original PGMA, while new peaks appeared at 3310 and 1568 cm −1 corresponding to the -NH and -NH 2 stretching vibration, respectively [24]. These data indicated that the modification with ethylenediamine on the PGMA surface is successfully performed. In Figure 4C, the peak at 580 cm −1 is attributed to the Fe-O stretching vibration, indicating that the Fe 3 O 4 has successfully deposited in PGMA microspheres via in situ deposition [23].   [25]. The pattern of the M-PGMA/PHGH microspheres shows a broad band appearing in the range from 15° to 25°, which is derived from the amorphous PGMA component and polymer shell [26]. No other impurities were observed on XRD patterns, suggesting that the as-synthesized microspheres were relatively pure. It is obvious that the characteristic diffraction peaks of Fe 3 O 4 are weakened in the pattern of the magnetic M-PGMA/PHGH microspheres, because of the presence of the amorphous template. From XRD results, it can be concluded that the iron oxide nanoparticles are encapsulated by PGMA, which is in good agreement with the SEM and TEM results.

Characterization of the M-PGMA/PHGH-CdTe Microspheres

Magnetism Assessment of M-PGMA/PHGH-CdTe Microspheres

The magnetic hysteresis loop of the M-PGMA/PHGH-CdTe hybrid microspheres was taken at room temperature ( Figure 6). M-PGMA/PHGH-CdTe hybrid microspheres displayed a super-paramagnetic property, and the saturation magnetization value was found to be 4.608 emu·g −1 , which is sufficient for magnetic separation from water solution. The saturation magnetization value of the M-PGMA/PHGH-CdTe microspheres is less than that of the pure magnetic iron oxide nanoparticles (61.87 emu·g −1 ), which can be explained by considering the diamagnetic contribution of the PGMA template surrounding the magnetic nanoparticles [27]. M-PGMA/PHGH-CdTe hybrid microspheres were dispersed in water to study their magnetic separation behavior. Figure 7 exhibits that M-PGMA/PHGH-CdTe hybrid microspheres rapidly move toward the magnetic field direction in an aqueous solution under the external magnetic field in 30 s (from Figure 7A to Figure 7B). After the cancellation of the external magnetic field, M-PGMA/PHGH-CdTe hybrid microspheres evenly spread out in water again with bottle shaking (from Figure 7B to Figure 7A). In addition, introducing the magnetic separation can make the M-PGMA/PHGH-CdTe hybrid microspheres recyclable. These microspheres are potent biocidal agents, which can be easily separated and mechanically directed by applying a magnet. This is a utility when a particle-bound bacteria needs to be captured for environmental monitoring or the particles are to be directed specifically to a location of bacterial colonies, such as in a water treatment system and cooling devices and pipes. Thus, the recyclable antibacterial performance of M-PGMA/PHGH-CdTe hybrid microspheres is the crucial point of our study and will be taken into account in our consequent work for their practical application.

The Fluorescence Assessment of M-PGMA/PHGH-CdTe Microspheres

Introducing the quantum dots CdTe can provide the resultant hybrid microspheres fluorescent feature, which was confirmed through fluorescent assessment by dispersing the microspheres into aqueous solution [28]. M-PGMA/PHGH-CdTe hybrid microspheres render orange-red fluorescence under the irradiation of the ultraviolet lamps in Figure 8A and show green fluorescence under the digital fluorescence microscope in Figure 8B. The fluorescent property of resultant hybrid microspheres verified well that the quantum dot CdTe were firmly immobilized on M-PGMA/PHGH hybrid microsphere surface.

Antimicrobial Functions

The antimicrobial property of the resultant hybrid microspheres was examined against S. aureus, B. subtilis, P. aeruginosa and E. coli by using the MIC method. MIC is considered to be the lowest concentration that completely inhibits against agar plate comparing, disregarding singly colony or a faint haze caused by the inoculum [29]. The MIC values of M-PGMA/PHGH-CdTe microspheres against S. aureus, B. subtilis, P. aeruginosa and E. coli are shown in Table 1. The quantitative data in Table 1 have been repeated three times, and the same results were obtained. Hybrid microspheres have powerful antibacterial property against both Gram-positive bacteria and Gram-negative bacteria and the MIC values of 64 mg/mL, 500 μg/mL, 32 μg/mL and 16 μg/mL against E. coli, P. aeruginosa, S. aureus and B. subtilis, respectively. Guanidine compounds with a broad spectrum of activity against both Gram-positive and Gram-negative bacteria are mainly used as ideal antimicrobial agents. Among various types of guanidine compounds, polymer synthesized from hexamethylene and guanidine salt have been the most extensively investigated, due to high water solubility, wide spectrum antibacterial activity, excellent antibacterial efficiency and nontoxicity. Zhang et al. synthesized polyhexamethylene guanidine hydrochloride and polyhexamethylene biguanidine hydrochloride by melting polymerization, and the corresponding antibacterial assessments against bacteria and fungi were carried out as well [6]. Qian et al. fabricated modified guanidine polymers composed of PHGH and an epoxy group by using condensation polymerization. The dynamic antimicrobial process of the guanidine polymer and the morphological change of bacterial cells were found by an atomic force microscope (AFM) test [30].

Guan et al. grafted guanidine polymer PHGH onto the cellulose fibers by in situ graft copolymerization of glycidyl methacrylate-modified PHGH onto cellulose fibers. Through PHGH functionalization, the modified cellulose exhibited high antimicrobial activity against E. coli [7]. The hypothesis for this study is that the smaller the functionalized particle is, the more active it will be, for smaller particles possess a larger surface area and, thus, provide more functional sites to contact the bacteria, which results in the improved antibacterial efficiency. To improve the antibacterial effect, M-PGMA/PHGH-CdTe microspheres with a nanostructure were prepared in this work by immobilization of guanidine polymer PHGH into nano-scale M-PGMA, and the antimicrobial activities get a great improvement against both Gram-positive bacteria and Gram-negative bacteria compared with bulk powder counterparts.

Experimental Section

Materials

Guanidine hydrochloride was purchased from Tianjin Huadong Chemical Reagent Plant. GMA was obtained from Tianjin Guangfu Fine Chemical Research Institute. Ferric chloride (FeCl 3 ), potassium hydroxide (KOH), ethylenediamine, Ferrous chloride (FeCl 2 ·4H 2 O), epoxy chloropropane (ECH) and ammonium hydroxide (25 wt%) were purchased from Beijing Chemical Company. Azobisisobutyronitrile (AIBN) and polyvinyl pyrrolidone (PVP) were available from Tianjin Chemical Reagent Plant and Shanghai Chemical Reagent Plant, respectively. CdTe were synthesized according to the previous literature [31]. The other reagents were analytical grade and were used without any purification.

Characterization

The morphology and structure of the samples were characterized using a XL30 ESEM-FEG scanning electron microscope (SEM). The XRD patterns were obtained with a Siemens model D5000 diffractometer equipped with a copper anode producing X-rays with a wavelength of 1.5418 Å. Data was collected in continuous scan mode from 10° to 80° with a 0.02° sampling interval. Fourier transform infrared (FTIR) spectra were recorded by using a Thermo Nicolet (Woburn, MA, USA) Avatar 370 FTIR spectrometer. Magnetization curves as a function of magnetic field were measured at 298 K under a magnetic field up to 10 kOe. The fluorescence of the samples was characterized using a NIKON PE2000-u digital fluorescence microscope.

Preparation of PGMA Microspheres

About 0.15 g of PVP was added into a mixed solution containing 120 mL of ethanol and 15 mL of deionized water. The mixed solution was added into a 250 mL flask in a 50 °C water bath after complete dissolution. Then 0.12 g of AIBN was mixed with 5 mL of GMA and subsequently added into the flask; the temperature was increased to 70 °C, and the reaction was kept for 16 h. After the polymerization, the whole system was cooled down, and the obtained microsphere emulsion was centrifuged and washed several times with ethanol and water. At last, the product was freeze-dried and preserved.

Preparation of NH 2 -PGMA Microspheres

About 2 g of the PGMA were added to a mixture of 20 mL of deionized water and 20 mL of ethanediamine. The mixture was stirred vigorously at 80 °C for 12 h. The whole process described above was performed under the protection of N 2 , and the solution was kept stirring at a certain rate. The resultant microspheres were centrifuged and washed several times with ethanol and water, respectively, to remove the impurities and freeze-dried in a vacuum.

Preparation of M-PGMA Microspheres

About 1 g of NH 2 -PGMA was added into 20 mL deionized water and dispersed to emulsion at room temperature. Then, the emulsion was cooled down to 10 °C. FeCl 3 (162 mg), and FeCl 2 ·4H 2 O (63 mg) was mixed in 10 mL of water at 10 °C with stirring. The mixed solution was added into the dispersed NH 2 -PGMA emulsion, and the system was pumped into vacuum rapidly down to a pressure of 10 mm Hg for 20 min. Then, the system was restored to the atmosphere pressure. About 2 mL of NH 3 ·H 2 O (25 wt%) was added into the flask, and the temperature was raised to 80 °C. The reaction was kept for 30 min, and the obtained M-PGMA microsphere emulsion was centrifuged several times and dispersed with ethanol and water to remove the impurities and freeze-dried in a vacuum.

Preparation of M-PGMA/PHGH Microspheres

Immobilization of PHGH on M-PGMA microspheres was accomplished via a two-step processes, including epoxide modified PHGH and PHGH immobilization. First, a 5 g mount of PHGH was added into 50 mL of deionized water. ECH (1 g) was added dropwise into the mixture during the last 30 min and kept stirring for 4 h. Then 50 mL of deionized water was added, and the reaction was kept for another 6 h at 60 °C to obtain the epoxide modified PHGH. In the second step, the epoxide-modified PHGH was added into 50 mL of ethanol, and 0.2 g of KOH were added to tune pH = 8. Then, 1 g of M-PGMA was injected into the mixture for 6 h. The obtained M-PGMA/PGHG microspheres were centrifuged and washed several times with ethanol and water, respectively, to remove the impurities and dried in a vacuum oven at 50 °C.

Electrostatic Adsorption of Dot CdTe

Typically, 0.75 mg of M-PGMA/PHGH microspheres were added into a 1.5 mL aqueous CdTe quantum dot solution protected by mercaptosuccinic acid and mixed by sonication for 10 min. After centrifugation, the supernatant was removed, and the precipitate was washed with ethanol, which was dispersed and centrifuged several times to obtain the M-PGMA/PHGH-CdTe microspheres.

Antibacterial Assessment

Staphylococcus aureus (S. aureus), Bacillus subtilis (B. Subtilis), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) were used as model microorganisms to determine the antimicrobial properties of the samples. The minimum inhibition concentration (MIC) of SiO 2 -PS-CDMH nanoparticles was determined by the similar agar plate method. The sample concentration varied from 32, to 64, to 128, to 256, to 512 and to 1024 µg/mL. The culture of each bacterium was diluted by sterile distilled water to ca. 100 CFU/mL, and the inoculated plates were incubated at 37 °C for a contact time of 12 h.

Conclusions

In conclusion, the M-PGMA/PHGH-CdTe hybrid microspheres with the properties of magnetism, fluorescence and antibiosis were successfully synthesized. The magnetic PGMA microspheres were synthesized as support, and PHGH was grafted on the template surface. Finally, the quantum dot CdTe was adsorbed on the microsphere surface. Antibacterial tests revealed that the M-PGMA/PHGH-CdTe microspheres exhibited powerful antibacterial performance against both Gram-positive and Gram-negative bacteria. Magnetic measurement showed that these hybrid microspheres possessed a super-paramagnetic property, and the saturation magnetization value was found to be 4.608 emu·g −1 . Magnetic behavior can make these antibacterial microspheres structural antibacterial materials separable in a rapid and easy way. The resultant hybrid microspheres with the quantum dot CdTe have targeted positioning and biological monitoring functions. Introducing the magnetism and fluorescence in the antibacterial field in this study opens up the possibility of the extensive study of antibacterial materials, widening their potential applications in medical devices, healthcare products, water purification systems, hospitals, dental office equipment, food packaging, food storage, household sanitation, etc.

Figure 1 .
1Schematic illustration for the formation processes of the multifunctional M-poly(glycidyl methacrylate) (PGMA)/poly(haxemethylene guanidine hydrochloride) (PHGH)-CdTe hybrid microspheres.

Figure 2 .
2SEM images of hybrid microspheres: (A) PGMA; (B) NH 2 -PGMA; (C) M-PGMA.

Figure 3 .
3(A,B) TEM images of M-PGMA/PHGH-CdTe microspheres; (C) SEM images and (D) EDX spectrum of the M-PGMA/PHGH-CdTe microspheres.

Figure 3 .
3Cont. 2.4. FT-IR Analysis of PGMA, NH 2 -PGMA and M-PGMA Microspheres FTIR spectra were recorded and used to identify the formations of the PGMA, NH 2 -PGMA and M-PGMA microspheres in each synthetic route. As shown in

Figure 4 .
4FTIR spectra of (A) PGMA; (B) NH 2 -PGMA; (C) M-PGMA hybrid microspheres.

2. 5 .
5XRD Analysis of M-PGMA/PHGH Microspheres XRD patterns of the magnetic M-PGMA/PHGH microspheres (Figure 5) show characteristic (220), (311), (400), (422), (511) and (440) peaks of cubic inverse spinel structure, which matched well with standard Fe 3 O 4 nanoparticles

Figure 5 .
5XRD patterns of the M-PGMA/PHGH hybrid microspheres. (a.u. = arbitrary unit).

Figure 6 .
6The magnetic hysteresis loop of the M-PGMA/PHGH-CdTe microspheres at 298 K.

Figure 7 .
7The photograph of the magnetic M-PGMA/PHGH-CdTe microspheres dispersed in aqueous solution without and with an external magnetic field.

Figure 8 .
8(A) The photograph of composite microspheres dispersed in water under ultraviolet irradiation; (B) Digital fluorescence microscope image of composite microspheres dispersed in water.

Table 1 .
1MIC Values of M-PGMA/PHGH-CdTe microspheres Against E. coli, 25922, P. aeruginosa, 27853, S. aureus, 25923 and B. subtilis, 6633.Antibacterial
Result

Gram-negative
Gram-positive
E. coli
ATCC 25922

P. aeruginosa
ATCC 27853

S. aureus
ATCC 25923

B. subtilis
ATCC 6633
MIC (μg/mL)
64
500
32
16

© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
AcknowledgmentsWe thank the National Natural Science Foundation of China (No. 50673033) and the National Natural Science Foundation of Jilin Province (No. 201115011) for the financial support of this research.Conflict of interestThe authors declare no conflict of interest.
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The IronChip evaluation package: a package of perl modules for robust analysis of custom microarrays

Yevhen Vainshtein
Mayka Sanchez
Alvis Brazma
Matthias W Hentze
Thomas Dandekar
Martina U Muckenthaler
The IronChip evaluation package: a package of perl modules for robust analysis of custom microarrays
S O F T W A R E Open Access
Background: Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Results: The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls. Conclusions: ICEP is a stand-alone Windows application to obtain optimal data quality from custom-designed microarrays and is freely available here (see "Additional Files" section) and at: http://www.alice-dsl.net/evgeniy. vainshtein/ICEP/

Background DNA microarrays are a popular high throughput technique to perform genome-wide molecular and genetic experiments. The method requires computational postprocessing of primary data, which can be a challenging task due to high data variability. Variability results from each of the many steps involved in such an experiment [1]. Computational data processing is required for image processing, extraction of raw data, storage and normalization of the raw data, feature extraction, final data analysis and biological interpretation of the results. Several software packages are available to perform the described tasks but it is frequently necessary to develop custom-made solutions to fulfil individual requirements and appropriate statistical evaluation of gene array data [2,3].

Experiments on whole-genome arrays, such as Affymetrix GeneChips are highly reproducible, but still are unable to provide reliable data for all genes, especially for those with low expression levels. Moreover, there are technical limitations on array design. For example, standard glass slide arrays can not accommodate more than 60,000 spots, including all controls and replicates. This is often not sufficient for complex genomes such as the human genome. The relationship between fluorescent signal intensity and gene expression levels is linear only for a certain range of concentrations of spotted material. Thus differences in the linearity range can become more pronounced for larger wholegenome arrays [4]. In contrast, customized microarrays contain a selection of genes. This allows to include more replicates and controls. Smaller gene numbers, and higher numbers of repetitions will increase reliability of the data obtained from each microarray experiment, especially for those genes that are expressed to low levels.

IronChip is a cDNA microarray platform specifically designed to analyze genes related to iron metabolism. We have developed two types of this platform to analyze both human and mouse genes [5,6]. The design of this microarray enables detection of small but physiologically significant changes in gene expression, due to the high number of repetitive features. The current version of the mouse IronChip contains 520 genes involved in iron homeostasis and related pathways. To improve array sensitivity and data robustness, each gene on the array is represented by several ESTs. Each EST, in turn, is represented by a minimum of six spots. Some of the most relevant iron-related genes are represented by up to 24 spots. This microarray further contains a collection of negative controls, specificity controls and positive (spike-in) controls [6]. Custom microarray platforms, such as the IronChip, provide more data than required or exploited in standard statistical analysis. To incorporate all the advantages such a chip design offers for data analysis we developed the IronChip Evaluation Package (ICEP). ICEP makes use of the high number of repetitions to improve data quality. The comparison of different ESTs enables reliable detection of transcript-specific regulation (e.g. alternative splicing variants of the same gene). Analyses of the positive and negative controls allow precise calculation of a reliable ratio cutoff as well as to estimate background noise, respectively. Implementation ICEP exploits a collection of Perl programs and utilities with a Perl Tk GUI (graphical user interface). The Perl routines were all newly custom written for the purpose of rapid and solid microarray data analysis. ICEP features a decision-tree based algorithm to optimize spot selection and exploit here in particular multiple repetitions of ESTs. ICEP applies grouping rules in its decision tree algorithm to calculate signal intensity ratios for each individual group of ESTs representing the same transcript (this is explained step by step in the application of the analysis pipeline together with supporting online material http://www.alice-dsl.net/evgeniy.vainshtein/ ICEP/ICEP_manual.html). The pipeline is summarized in Figure 1A. ICEP does not use or require any existing software libraries and it can directly process simple tab delimited tables of array data of any type. It adds its optimized spot selection, filtering and normalization procedure to standard software such as Bioconductor [3] and can used in combination with these or equally well alone.  [8]. ICEP uses these output files (generic tab-delimited text tables containing the normalized signal intensity and background data) for further analysis.

ICEP is packaged to a Windows executable with a PDK (Perl developer kit). It can be operated in a command line mode or in a batch mode for the analysis of multiple arrays. A simple editor allows to specify all microarray data files for batch analysis. In a command line mode the ICEP analysis core itself can be executed under any operating system supporting Perl. Some Windows specific features, such as exporting of output data to an Excel table or Perl Tk GUI requires adaptation to a specific operating system. In general, the modular structure of ICEP allows porting it to any other operating systems supporting Perl.

Data formats

ICEP recognizes any generic tab-delimited text tables from any type of gene microarray containing the normalized signal intensities and background data (e.g. from the ChipSkipper application; [7]). Chip-Skipper generates a tab-delimited text table containing raw and normalized signal intensity values, background signal intensity, physical coordinates of a spot relative to an upper left corner of a glass slide, cDNA sample position on original PCR spotting plate (row, column, plate Nr), some flags and statistical values related to the spot geometry and other internal values. The ICEP uses only few of those columns: spot and clone coordinates, comments and background-compensated and normalized signal intensity value from both channels. The build-in utility recognizes not only different formats of a Chip-Skipper output file, but any generic tab-delimited text file gene expression array data can be processed by ICEP using the provided flexible configuration tool and alternative input file formats are added using the provided flexible configuration tool. Results are saved in tab-delimited format or Microsoft Office Excel formats.

Performance

We tested ICEP performance by measuring time consumption to analyze microarray data from different mouse IronChip versions (version 2.0 contains 559 transcripts, while version 7.0 contains 932 transcripts) (Figure 2A) or by analyzing a set of virtual arrays (1000 to 9000 features, with 1000 features step). On average, ICEP could evaluate 208 features per second. The time per run increases linearly with an increasing number of analyzed features ( Figure 2B).

Results and Discussion

User interface ICEP has been developed as a stand-alone application and does not require any special environment. It runs under any Windows operating system (it was tested on Windows 2000, XP and Vista). The interface has been designed to be highly user-friendly and interactive. It helps the user to apply all analyses while hiding the complexity of the underlying statistical methods. The interface provides easy access to different layers of microarray analysis, including single array analysis, dyeswap analysis and the generation of a final report. A step by step user manual is available in additional file 1 and at: http://www.alice-dsl.net/evgeniy.vainshtein/ICEP/ ICEP_download.html

Application of the analysis pipeline

The ICEP data analysis package was designed to be both highly flexible and user friendly. Data analysis involves an analysis pipeline that is composed of three elements: single array analysis, dye-swap experiment analysis and final report generation ( Figure 1B). In addition, the pipeline can either be executed directly or in a batch analysis mode.

In our application example, we utilized the mouse IronChip microarray that contains 520 genes as well as positive and negative controls. The controls are represented by 1400 spike-in control spots (positive controls) and 2400 background control spots (negative and specificity controls). 520 genes are represented by 880 ESTs. Genes are represented by 2 or more ESTs. Each EST was spotted on the array at least 6 times. 5400 spots in total are located on the array.

At the first level of analysis (single feature level), ICEP performs logarithmical transformation of the data and separates background and control spots from the rest of the data. ICEP then calculates a background cut-off value based on median signal intensities of all background and negative control spots, and an intensity ratio cut-off value, based on signals from the spike-in controls. Intensity ratios of all remaining genes are calculated as well. At the same time, ICEP performs a feature extraction procedure, whereby all repetitive features, representing the same EST are grouped together. After calculating the background and ratio cut-off values ICEP assigns the following flags: (1) the P-call flag (true positive call), which is based on a comparison of a signal intensity of each channel with the background cut-off value (Table 1); (2) the regulation flag (significant difference in gene expression between two channels), which is based on comparison of a signal ratio to the ratio cut-off value ( Table 2). At the second level of analysis (EST level) ICEP assigns further flags to ESTs and estimates the data quality based on flags recorded on a single feature level. EST P-call flags are calculated by ICEP according to the rules given in Table  3. Definition of a P-call flag (at the EST level) is based on the P-calls of individual features. Corresponding threshold is set to 60%, due to the fact that a control microarray experiment (hybridization of a Hemin-and Desferrioxamine-treated HeLa cells) shows similar results to published data only when the EST P-call threshold is about 60%. Significant increase of the EST P-call threshold causes additional false negative results while decreasing of this value causes additional false positive results

The comparison of average or median signal intensity ratios to the previously calculated ratio cut-off value yields UP/DOWN/NONE-flags, similar to the flag calculations described above. At the EST level ICEP calculates the relative error: the ratio between the standard deviation and the average of signal intensity ratios of all features representing single ESTs. At the transcript level ICEP uses the relative error as a measure of reliability of technical and biological replicates.

Preceding transcript level analysis, ICEP analyzes whether any bias has occurred as a consequence of the dye (Cy5 or Cy3 labelled nucleotides) incorporated into hybridization probes. To avoid such dye bias in two-colour microarray hybridizations the experimental and the control sample are routinely labelled with Cy5 and Cy3     labelled nucleotides, respectively, plus the other way around (dye swap). Such analysis avoids inconsistent signal intensity ratios that are artefacts due to the dye incorporated into the hybridization probe. Depending on whether an EST shows a similar average signal intensity ratio within the dye-swap data set, ICEP defines a dye-swap reliability flag (Table 4). On the transcript level ICEP applies grouping rules to calculate signal intensity ratios for a group of ESTs representing the same transcript. For this purpose ICEP is using all quality flags described before. On this level ICEP decides whether to average signal intensity ratios from different ESTs to a single value, to treat each EST as a separate transcript or to mark the complete set of ESTs as non-reliable. ICEP is able to analyze and group values from up to six similar ESTs representing a single gene (six is the maximum value in the current version of the IronChip microarray). To illustrate the grouping procedure a scheme is presented in Figure 3 which is based on 2 ESTs representing a single gene. Table 5 represents the possible flag combinations.

Validation

We applied the ICEP software to analyze IronChip microarray data. We selected a previously reported experiment that analyzes iron-loaded (hemin-treated) and iron-deficient (Desferrioxamine-treated) HeLa cells. Cellular iron overload or deficiency caused the expected changes in gene expression [6,8]. Table 6 represents the data of a dye-swap experiment. Application of ICEP reveals the previously reported and experimentally validated changes in mRNA expression of genes such as Tfrc (Transferrin receptor 1), Slc11a2 (NRAMP2; DMT1: Natural resistance-associated macrophage protein 2; Divalent metal transporter 1) and Ftl (Ferritin light chain; Ferritin L subunit).

The complete data set (additional file 2) and ICEP installation package (additional file 3) are available in the "Additional Files" section and on-line at the web page.

Comparison to other microarrays analysis software

We compared the performance of ICEP to other genome array tools such as Bioconductor [3], Gene-Spring (Agilent, Santa Clara, CA) [9] and a web-based tool called "Expression Profiler" [10]. While GeneSpring EST shows "P" or "M" p-calls and is UP-regulated in the Cy5 and DOWN-regulated in the Cy3 experiment, or vice versa.

TRUE

EST shows "P" p-call and is UP-regulated or DOWNregulated in the, Cy5 while the Cy3 experiment shows a tendency towards the correct direction based on the ratio cut-off value, or vice versa TRUE EST shows "P" or "M" p-call, but both experiments show NONE-regulated with one is UP-regulated or DOWNregulated other is NONE-regulated, a tendency of regulation towards the correct direction based on a ratio cut-off ICEP determines a reliability flag by evaluating the P-call and regulation flags of ESTs. The reliability flag is used by ICEP to distinguish reliable from unreliable expression changes. contains a big collection of array analysis tools it is inferior in the processing speed. Bioconductor is faster than ICEP (about 20% in running time) but as well as GeneSpring by default has limitations in processing multiple repetition features compared to ICEP. Using Bioconductor and R programming language it is possible to implement any feature recognition and statistical algorithm, but this requires additional coding work and tests. Expression profiler provides a very convenient interface and world-wide access to data analysis, but has a limited amount of statistical instruments due to its web-based nature. In general, most of the available software does not offer appropriate feature extraction from different multiple spotting regimes or recognition of feature groups. GeneSpring and Bioconductor support averaging of replicated features but do not support grouping rules for the analysis of repetitive ESTs.

Conclusion

We introduce a new flexible microarray analysis tool named ICEP, optimized for robust statistical analysis of specialized custom cDNA or oligonucleotide microarrays. Our analysis yields values for ratio cut-off, background noise, multiple repetitions and detailed feature extraction as well as grouping rules. ICEP is easily extended to support further input and output data formats and different data transformation steps can be added. ICEP allows rapid post-processing of microarray data on a user-friendly platform. Software, example data and a tutorial are open source, free and available for downloading.

Availability and requirements

Figure 1
1IronChip analysis work flow. (A) Flow chart of ICEP data processing and evaluation. Data evaluation with ICEP is organized into three functional modules: Single feature, EST and transcript evaluation. (B) In our application example, hybridized microarrays were scanned on a GenePix 4000B Microarray Scanner (Axon Instruments, Union City, CA, USA) and processed (feature background subtraction and normalization) by the ChipSkipper software

Figure 2
2ICEP run time chart. (A) Run time analysis of different microarray versions containing an increasing number of features. The plot shows the resulting increase in ICEP run time for different IronChip versions. (B) A set of virtual arrays of 1000 to 9000 features was analyzed. We used a general tab delimited format. Robust statistical analysis included analysis of background noise, ratio cut-off, evaluation of multiple repetitions, detailed feature extraction and grouping results. ICEP Run time increases linearly with the increase of the total number of analyzed features. On average, ICEP evaluates 208 features per second.

intensity is higher than the background cut-off value in both channelsMThe signal intensity is higher than the background cut off value in one channel and lower in the other channel A The signal intensity is lower than the background cut off in both channelsDefinition of a P-call flag (at the feature level) to distinguish bona fide signals from background noise

not apply and more than 60% of features representing one EST have the p-calls "M" and "P" than assign an EST P-call "M" apply and more than 60% of features representing one EST have the p-calls "M" and "A" than assign a EST P-call "A"Definition of a P-call flag (at the EST level) is based on the P-calls of individual features and allows to distinguish UP-/DOWN-/NONE-

Figure 3
3Schema of grouping two ESTs. The flow diagram indicates different decision steps in the analysis tree. The grouping procedure for three and more ESTs is done similarly, using rule 1 for 2 ESTs recurrently.

Table 1 P
1-call definition

Table 2
2Regulation flag definitionRegulation
flag

Conditions

UP
Ratio between signal intensities is higher than
corresponding ratio cut-off

DOWN
Ratio between signal intensities is lower than
corresponding ratio cut-off

NONE
Ratio between signal intensities is in between upper and
lower ratio cut-off

Definition of a regulation flag (at the EST and the transcript level) to
distinguish UP-/DOWN-/NONE-regulated ESTs and transcripts from each other

Table 3
3EST P-call definitionRule
Nr.

EST
P-call

Conditions
Description

1
P
P≥ 60% ⇒ P
If more than 60% of features
representing one EST have a p-call
"P" then assign an EST P-call "P"

2
M
M≥ 60% ⇒ M
If more than 60% of features
representing one EST have a p-call
"M" then assign an EST P-call "M"

3
A
A≥ 60% ⇒ A
If more than 60% of features
representing one EST have a p-call
"A" then assign an EST P-call "A"

Table 4
4Dye-swap reliability flag definitionDye-swap
flag

Conditions

Absent
EST shows the p-call "A" in the Cy5 and Cy3 experiment

Non reliable The Cy5 and the Cy3 experiment show identical
regulatory behavior (both UP, both DOWN)

Non
regulated

EST does not show any regulation in both experiments

TRUE

Table 5
5The EST grouping rules represent rules for a decision-tree based algorithm to summarize expression data from several ESTs, representing one transcript. These are contained within a core table.ESTs grouping rules (2 ESTs grouping)

EST 1
EST 2
Rel. Error
Transcript

DS flag
Regulation
DS flag
Regulation
Flag
Value
Regulation

absent
N.A.
absent
N.A.
absent
0
NONE

non regulated
NONE
non reliable
0
NONE

non reliable
N.A.
non reliable
0
NONE

TRUE
UP/DOWN
non reliable
Average
NONE

non regulated
NONE
non regulated
NONE
non regulated
Average
NONE

non reliable
N.A.
non reliable
0
NONE

TRUE
UP/DOWN
non reliable
Average
NONE

non reliable
N.A.
non reliable
N.A.
non reliable
0
NONE

TRUE
UP/DOWN
non reliable
Average
NONE

TRUE
UP
TRUE
UP
≥15
non reliable
Average
UP

UP
≤15
TRUE
Average
UP

TRUE
DOWN
non reliable
Average
NONE

TRUE
DOWN
TRUE
DOWN
≥15
non reliable
Average
DOWN

DOWN
≤15
TRUE
Average
DOWN
UP
non reliable
Average
NONE

Table 6
6Expression increase in iron-loaded and irondeficient cellsGene name Regulation (D/H) Final flag Ratio(av) P-call (av)Thetable represents the average ratios of differentially expressed genes in Hemin-and Desferrioxamine-treated HeLa cells. The relative errors are shown. The table contains only selected columns and genes. The full table is attached (see Additional files 1) Additional file 1: ICEP user manual. ICEP manual.pdf file contains a local copy of a user manual web page http://www.alice-dsl.net/evgeniy. vainshtein/ICEP/ICEP_manual.html. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2105-11-112-S1.PDF ] Additional file 2: ICEP installation package. ICEP.MSI file contains installation logic and all necessary files to install ICEP on any Windows operating system. Windows Installer is required to execute MSI package. In the case it is not present on the system, one could download it from Microsoft web page. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2105-11-112-S2.MSI ] Additional file 3: Example dataset and ICEP analysis results. This ZIP archive includes several files containing a) 4 input data tables, corresponding to 2 IronChip hybridization experiments with a dye swap; b) 2 intermediate results files with single array evaluations; and c) Excel file with final results. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2105-11-112-S3.ZIP ] Abbreviations Cy3/Cy5: reactive water-soluble fluorescent dyes of the cyanine dye family. Cy3 dyes are yellow-orange (550 nm excitation, 570 nm emission), while Cy5 is fluorescent in the red region (650/670 nm); EST: Expressed Sequence Tags -ESTs are small pieces of DNA sequence (usually 200 to 500 nucleotides long) that are generated by sequencing either one or both ends of an expressed gene; GUI: Graphical User Interface; ICEP: IronChip Evaluation Package; PDK: Perl developer kit. Gene names TFRC: Transferrin receptor 1 MT2A: Metallothionein 2 EPAS1: Endothelial PAS domain-containing protein 1; Hypoxia-inducible factor 2 alpha (HIF-2 alpha) SLC11A2: Solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2; Natural resistance-associated macrophage protein 2 (NRAMP2); Divalent metal transporter 1 (DMT1) ACTB: Actin, beta, cytoplasmic ALAS2: Aminolevulinic acid synthase 2, erythroid FTL: Ferritin light chain; Ferritin L subunit HSPH1: Heat shock 105 kDa/110 kDa protein 1 HMOX1: Heme oxygenase (decycling) 1 HSPA1L: Heat shock 70 kDa protein 1-like HSPA1A: Heat shock 70 kDa protein 1A.TFRC
UP
TRUE
5, 23 ± 0, 22 P
MT2A
UP
TRUE
1, 9 ± 0, 69
P

EPAS1
UP
TRUE
1, 76 ± 0, 1
P

SLC11A2
UP
TRUE
1, 57 ± 0, 24 P

ACTB
UP
TRUE
1, 47 ± 0, 29 P

ALAS2
UP
TRUE
1, 46 ± 0, 11 P

FTL
DOWN
TRUE
-1, 58 ± 0, 06 P

HSPH1
DOWN
TRUE
-2, 28 ± 0, 13 P

HMOX1
DOWN
TRUE
-3, 17 ± 0, 16 P
HSPA1L
DOWN
TRUE
-3, 8 ± 0, 46 P

HSPA1A
DOWN
TRUE
-5, 3 ± 0, 03 P

Vainshtein et al. BMC Bioinformatics 2010, 11:112 http://www.biomedcentral.com/1471-2105/11/112 Page 3 of 7
AcknowledgementsWe thank members of the Hentze group, members of the EMBL Genomics Core Facility and members of Molecular Medicine Partnership Unit for support with the IronChip platform and useful discussions.Submit your next manuscript to BioMed Central and take full advantage of:
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Virology Journal Barriers to coliphage infection of commensal intestinal flora of laboratory mice

Laura M Kasman firstname.lastname@example.com
Department of Microbiology and Immunology
Medical University of South Carolina
BSB-201, 173 Ashley AvenueP.O. Box 25050429403CharlestonSCUSA

Virology Journal Barriers to coliphage infection of commensal intestinal flora of laboratory mice
10.1186/1743-422X-2-34BioMed Central Page 1 of 7 (page number not for citation purposes) Research * Corresponding author
Background: Growth characteristics of coliphage viruses indicate that they are adapted to live with their Eschericia coli hosts in the intestinal tract. However, coliphage experimentally introduced by ingestion persist only transiently if at all in the gut of humans and other animals. This study attempted to identify the barriers to long term establishment of exogenous coliphage in the gastrointestinal (GI) tracts of laboratory mice. Intestinal contents were screened for the presence of coliphage and host bacteria, and strains of E. coli bacteria from different segments of the GI tract were tested for susceptibility to six common laboratory coliphages.Results: Contrary to expectations, coliphage were not evident in the GI tracts of laboratory mice, although they were occasionally detected in feces. Commensal flora showed extreme variability within groups of mice despite identical handling and diet. Less than 20% of 48 mice tested carried E. coli in their gut, and of 22 commensal E. coli strains isolated and tested, 59% were completely resistant to infection by lambda, M13, P1, T4, T7, and PhiX174 coliphage. Lysogeny could not be demonstrated in the commensal strains as mitomycin C failed to induce detectable phage. Preexisting immunity to phages was not evident as sera and fecal washes did not contain significant antibody titers to six laboratory phage types.Conclusion:Lack of sufficient susceptible host bacteria seems to be the most likely barrier to establishment of new coliphage infections in the mouse gut.

Background

Coliphage have traditionally been isolated from sewage, where they arrived, presumably, after passing through the GI tracts of animals inhabited by commensal coliform bacteria. However, information on the interaction of natural coliphage with the commensal flora of the GI tract in situ is sparse. The results of ingestion of defined high titer bacteriophage preparations by laboratory animals or by humans have been described in many previous studies (reviewed in [1]). Although there is evidence in some reports of coliphage replication in the gut, the phage infections are consistently transient, becoming undetectable in 3-10 days [2][3][4]. Notable exceptions are cases of gnotobiotic mice inoculated with defined phage-host systems, in which phage and host populations in feces were detectable for up to 98 days [4]. However, in animals with complex, established gastrointestinal microflora, observations concur that exogenous phage do not establish sustained productive infections of the commensal bacteria. The nature of this apparent barrier to persistent bacteriophage infection of the normal GI tract is of practical interest since as parasites of commensal bacteria, bacteriophage have the potential to impact health by altering the GI flora. Conversely, it may be possible to engineer phage specifically to alter the commensal flora in situ for therapeutic benefit.

Previous work has already shown that potential physical obstacles to bacteriophage infection of bacteria in the gut are not significant. The acid environment of the stomach would at first appear to be an obvious barrier, but coliphage have been shown to maintain their infectivity when passed through the stomach or exposed to gastric fluids [4][5][6][7]. Attachment of phage to the host bacteria could be inhibited by secretions or deficiencies of cofactors in the GI environment, however infection of bacteria in the gut has also been demonstrated for a variety of phage [5,8,9], including the temperate phage, CTXphi, of Vibrio cholerae. CTXphi in fact infects its host more efficiently in vivo than in vitro, because the phage has adapted to use as its receptor V. cholerae surface molecules expressed only when the bacteria colonizes the gut [10]. However, V. cholerae is not normal flora, and CTXphi lysogens and phage are cleared in the usual time frame. In addition, antimicrobial phage therapy trials have demonstrated phage infection of bacteria in the peritoneal cavity, blood, muscle [1,11] and embryonated hen eggs (L. Kasman, unpublished observations). Therefore, the in vivo environment itself does not directly prevent infection of bacteria by phage.

Phage are immunogenic, even when introduced into the GI tract [5,9] and so anti-phage antibodies may play a role in the barrier to new phage infections in commensal coliforms. Alternatively, susceptible host bacteria may not be present at high enough densities to allow persistent infection by coliphage. When phage concentration are low, very high densities of host cells can be required to enable a detectable number of infections to occur [12]. Commensal intestinal bacteria are a climax community in which an estimated 400 different species of bacteria fill all niches with regard to locations and energy sources, so that newcomers find it difficult or impossible to become established [13,14]. A new bacteriophage could in theory face a similar obstacle, although not in terms of metabolic needs, as most phage lysogens inhibit superinfection of their host by similar phage types.

In this study, these potential barriers phage colonization of commensal bacteria were assessed for E. coli, by testing the gut contents and feces of several strains of laboratory mice for the existence of free coliphage and E. coli capable of supporting phage infection. In addition, six well characterized coliphage types were used to test commensal E coli bacteria for susceptibility to phage infection, and sera and mucosal secretions were examined for evidence of anti-coliphage antibodies.

Results

Screening for endogenous coliphage in the feces of laboratory mice

Feces of two independent groups of 40 and 48 ICR mice, were screened every three days for a total of four samplings for the presence of naturally occurring (endogenous) coliphage. Fresh fecal pellets were collected for each mouse individually, resuspended in LB, then centrifuged and the supernatants spotted onto indicator E. coli in top agar within 6 h of collection. Feces of small groups of C57BL/6 and nude mice were also collected such that over 400 samples were screened on indicator E. coli strains CN13 (F-) and ER2738 (F+) for somatic and male-specific phages respectively. A total of three samples from two mice were positive for phage on the somatic phage host only. All other feces samples were negative, indicating that even in the mice positive for phage, the phage could only be detected transiently.

Screening for coliphage and lysogens in the murine intestinal tract

In order to investigate coliphage prevalence in the GI tract as well as feces, 48 ICR and 15 C57BL/6 mice were euthanized after a final feces collection, and GI tracts removed aseptically to individual sterile petri dishes. Each GI tract was divided into four segments: (1) stomach and duodenum, (2) 10 cm of small intestine midway between the duodenum and cecum, (3) cecum, and (4) colon and rectum. Intestinal contents were collected by flushing each segment with 1 ml L-Broth containing CaCl 2 and MgSO 4 , into a microfuge tube. Samples were gently vortexed, subjected to low speed centrifugation to pellet large particulates, and the supernatants retained for phage detection as described below. Known amounts of T4 and M13 phage were added to rinses of one mouse as a control, which demonstrated that the collection procedure and centrifugation did not remove or destroy phage (data not shown).

Due to the near absence of detectable phage in ICR mouse feces initially collected, two strategies to enhance coliphage detection were applied to the supernatants from the murine GI tracts and final feces collection from ICR, C57BL/6 and nude mice. First, in order to amplify phage which might be present at levels too low to detect, permissive E. coli host strains ER2738 or CN-13 were added to liquid cultures containing aliquots of gut or fecal supernatants. Second, mitomycin C was added since coliphage lysogens, which are otherwise undetectable by plaque assay, can usually be reactivated to the free state by treatment of the bacteria with the chemical mitomycin C. Combinations of permissive cells and mitomycin C produced five different culture conditions for the enrichment of phage recovery from feces and gut contents (Table 1). Supernatants from overnight cultures under these conditions were assayed for the presence of phage by spotting on ER2738 and CN13 agar overlays. Table 1 summarizes the results of several experiments. No male-specific bacteriophage were detected in gut contents or feces of any laboratory mice from two suppliers and three animal strains (nude mice were analyzed by feces only). Somatic bacteriophage were detected in the feces but not the gut contents of one ICR mouse after enrichment, regardless of mitomycin C treatment. Mitomycin C failed to produce any more detectable coliphage.

Prevalence of commensal E. coli in laboratory mice

Prevalence of commensal E. coli was studied in 48 sixweek old female ICR mice, shipped together and housed in 12 groups of 4 mice for two weeks in the MUSC animal facility, maintained on a diet of tap water and sterilized pellets. Aliquots of fecal suspensions and intestinal segment rinses for all mice were plated on individual MaConkey agar plates. Although large numbers of bacteria grew from all of the samples except some duodenum segments, less than half of the plates had colonies with morphology and color consistent with E. coli. Sixty-four individual colonies were chosen from 64 different plates and typed using the BioMerieux api20E system. It was found that only 20 of the 64 selected colonies were E. coli, 36 were Klebsiella teragina or Enterobacter aerogenes, 3 were Klebsiella ornithinolytica, 1 was Klebsiella pneumoniae pneumoniae, 1 was Myroides chryseobacterium indologenes, and 3 could not be identified. The 20 E. coli isolates were from 8 mice, so that only 8 of 48, or 17%, of the mice were found to carry E. coli. E. coli prevalence and diversity of commensal flora was similar in subsequent smaller groups of mice surveyed.

Susceptibility of commensal E. coli to laboratory-adapted coliphage

In total, 31 commensal E. coli strains recovered from four different gastrointestinal compartments and verified by BioMerieux species identification were collected from both ICR and C57BL/6 mice. These strains were tested for susceptibility to six well-characterized laboratory adapted coliphage (Lambda, M13, P1, øX174, T4, and T7) by spotting of two dilutions of each phage onto an agar overlay inoculated with the strain to be tested. Divalent cations CaCl 2 and MgSO 4 were supplied in the overlay as they are required for infection by P1. Susceptibility was observed as confluent lysis or individual plaques in the bacterial lawn after 18 h incubation. Isolates from different compartments of the same mouse that had identical phage susceptibility profiles were considered to be the same strain, reducing the number of unique isolates from 31 to 22. Less than 50% of these commensal strains were susceptible to any of the six coliphage ( Table 2). None of the 22 strains were susceptible to M13mp18 or lambda phage. P1 was the broadest host range phage, and was able to lyse 8 of 22 strains (36%) although plaques were considerably smaller in all cases compared to T4, T7 or øX174. Similar resistance was found in a panel of 15 human clinical E. coli isolates tested for susceptibility in the same manner ( Table 2).

Screening of mouse serum and mucosal secretions for anticoliphage antibodies

Pre-existing immunity in the form of mucosal or serum antibodies might account for the absence of coliphage in the gut. Fecal washes from 22 mice positive for lactose fermenting commensals were screened for IgA antibodies against M13, Lambda, P1, PhiX174, T4 or T7 phages by ELISA. There was variability in IgA reactivity between
- - ICR GI 1 - - - - - C57BL/6 GI 1 - - - - -
1 All four compartments were tested separately 2 -represents none detected. Minimum detectable titer was 10 2 /ml before enrichment. 3 Average of two mice, out of 88 tested. All others were negative.

mice, but each individual mouse had similar reactivity to all 6 phages ( Figure 1A). Based on a cut-off of two standard deviations from the mean IgA response for any given mouse, there was no evidence of phage specific mucosal IgA antibodies in this group of animals. Sera from 9 mice verified to be carriers of E. coli were likewise assayed for the presence of IgG and IgM antibodies. No evidence of phage-specific serum antibodies were found in any of the mice (Fig 1B)

Discussion

Bacteriophages are assumed to be a normal component of mammalian gastrointestinal microbial flora since they are commonly isolated from feces and raw sewage and grow most efficiently at temperatures approximating mammalian body temperature. This study focused on the bacteriophages of E. coli, since it is the commensal bacterial species for which the most characterized phages are known and it is easily grown in vitro. We also primarily used outbred mice (strain ICR) to more closely approximate a human population, although diet and environment were kept homogeneous. Contrary to expectations, with the exception of transient somatic phage infections in the feces of 2 of 88 mice, coliphage were not evident in the GI tracts of laboratory mice examined in this study. Our detection limit was 100 PFU/ml for GI tract rinses and approximately 100 PFU/gram feces before enrichment. However, in theory, a single infectious phage particle or single lysogenic cell should be detectable after enrichment. Coliphage densities in feces of domestic animals have been reported to vary widely by species [15]. Our results suggest that laboratory mice have some of the lowest fecal phage densities tested for domestic animals, but comparable to that reported for humans [15]. This was true for mice from two suppliers, and could have been a result of intentional association of the laboratory animals with a defined flora at the breeding facility. However, a defined flora also was not evident in these animals. At least with regard to aerobes, the commensal flora of the mice was very diverse despite the fact that the animals were all maintained in a barrier facility and fed the identical sterilized chow and water. E. coli, in particular, were detected in less than 20% of the animals, and 54% of animals produced no lactose fermenters at all on MaConkey agar.

The finding that murine commensal E. coli strains isolated were mostly resistant to infection by lambda, M13, P1, T4, T7, and PhiX174 coliphage was at first suggestive of a climax community of phage. However, superinfection exclusion became an unlikely mechanism for this resistance given the lack of phage production in response to mitomycin C. In addition, no phage-specific antibodies to known coliphage types were found in the sera or fecal washes of mice known to be colonized with E. coli. Phage are well known to be highly immunogenic in mice when injected and to pass into the blood when ingested in large numbers [7]. Taken together, these results indicate that the inability to establish persistent coliphage infections in commensal flora was not due to a climax community of phage already existing in the mouse.

Given the small percentage of commensal E. coli strains found to be susceptible to known coliphage types, the main barrier to establishment of new coliphage infections in the gut was most likely insufficient host cell density. It is estimated that in human E. coli carriers, 1 in 10,000 bacterial cells in the gut is an E. coli cell, and one gram of normal human feces contains roughly 10 6 live E. coli. If ideal conditions were present, such that there were 10 6 susceptible cells per ml of gut content, and the gut is assumed to approximate a liquid environment, then it can be calculated that a given phage present at a concentration of only 10 PFU/ml would be expected to infect on average two E. coli cells per ml of gut per hour [12]. However, conversely, in order to infect every available E. coli cell in such ideal conditions would require a phage concentration exceeding 1.5 × 10 6 PFU/ml. Based on our experience, the mouse intestinal volume is less than 3 mls, and the susceptible E. coli population is much lower. In addition, the majority of intestinal bacteria are located in the cecum and colon, where they will be eliminated from the body the most rapidly. It should be noted that the majority of microorganisms in the mammalian gut are anaerobes. Very little is known about bacteriophage of these organisms, but is possible that bacteriophages of anaerobes or bacteria other than E. coli would produce a different outcome.

Conclusion

The purpose of this investigation was to identify the barriers to long term establishment of coliphage in the GI tracts of laboratory mice. It was found that (1) laboratory Absence of specific antibodies to commensal coliphage by ELISA Figure 1 Absence of specific antibodies to commensal coliphage by ELISA. (A) Fecal washes from 22 ICR mice found to be colonized with lactose fermenting commensal bacteria were assayed in duplicate for IgA antibodies against M13mp18, Lambda, P1, øX174, T4 and T7 coliphages in parallel ELISA plates. Mice 2B, 2R, 2G, 2N, 3G, 3R, 4N, and 6G were eventually shown to carry E. coli. Each symbol represents the mean absorbance reading for one phage type. (B) Sera from 9 mice verified as E. coli carriers were assayed in duplicate for IgG and IgM antibodies against the same six coliphage by ELISA. Each symbol represents the mean absorbance reading for one phage type.

animals living under identical conditions and with an identical diet can have very different populations of commensal intestinal bacteria, (2) E. coli, while common in human intestinal tracts were relatively rare in the guts of laboratory mice, (3) commensal E. coli found in the GI tract of normal laboratory mice were resistant to several types of coliphage. In conclusion, lack of sufficient susceptible host bacteria seems to be the most likely barrier to establishment of new coliphage infections in the mammalian gut. The results suggest that altering commensal E. coli with coliphage in situ will be very difficult due to the high variability of commensal flora between individuals, and the low probability of a coliphage particle making contact with a susceptible cell in the gut.  [16], except that New England Biolabs strain ER2738 was substituted for strain F amp E. coli. Fecal washes were prepared by resuspending fecal pellets in PBS containing 0.2% sodium azide at a ratio of 100 mg feces per ml. Particulates were pelleted at 14 krpm in a microfuge and the supernatants frozen until analysis.

Methods

Bacterial strains and coliphage

Spotting Assay for presence of phage and analysis of phage susceptibility

The EPA protocol for detection of coliphage in drinking water [16] was followed except that E. coli strain ER2738 was substituted for strain F amp . Fecal pellets were resuspended as a 10% slurry (weight to volume) in LB containing 10 mM CaCl 2 and MgSO 4 . For detection of phage in feces, slurries were centrifuged 10 minutes at 14 Krpm in a microcentrifuge to remove particulates before spotting 10 µl onto E. coli inoculated top agar overlay. For determination of phage susceptibility, verified commensal E. coli strains or ER2738 and CN13 control strains were grown to logarithmic phase in LB and 0.1 ml of culture used to inoculate 3 mls molten LB top agar (0.6% agar, 42°C) which was poured onto an LB agar base. When top agars were dry, 10 µl intestinal wash or 5 µl of two dilutions of lab adapted phage (Lambda, M13mp18, P1, PhiX174, T4, and T7) were spotted onto the top agar. For phage susceptibility testing, the highest dilution producing complete clearing on the control strain, and the dilution ten-fold more concentrated were used. Plates were read after incubation 18-20 h at 37°C.

Bacterial species verification

For isolation and verification of commensal E. coli strains, single bacterial colonies with suspected E. coli morphology were picked from MaConkey agar plates inoculated with gut contents or feces. Each colony was restreaked on a separate MaConkey agar plates and single colonies analyzed with the BioMerieux api20e typing kit for species identification according to manufacturer's instructions (BioMerieux, Inc. Durham, NC).

Table 1 :
1Titers of bacteriophage after enrichment cultures from murine feces and contents of the GI tractEnrichment method

1
2
3
4
5

Mouse strain
source
No additions
CN-13 (F-)
bacteria

CN-13 (F-)
bacteria With
mitomycin C

ER2738(F+)
bacteria

ER2738(F+)
bacteria With
mitomycin C

ICR
feces
-2
7 × 10 5 PFU 3
6 × 10 5

Table 2 :
2Susceptibility of commensal E. coli strains to lysis by laboratory coliphage (percent susceptible strains)Source of E. coli
Lambda
M13
P1
øX174
T4
T7
none

Mouse feces and GI tracts (n = 22)
0 (0%)
0 (0%)
8 (36%)
3 (14%)
4 (18%)
3 (14%)
13 (59%)
Human clinical specimens (n=15)
1 (7%)
1 (7%)
6 (40%)
1 (7%)
1 (7%)
2 (13%)
7 (47%)

Difco) as noted. Mitomycin C was purchased from Sigma and used at a concentration of 2 µg/ml. Female ICR and C57BL/6 mice were obtained from Harlan Laboratories, C57BL/6 mice were also obtained from Jackson Laboratories. Feces from nude mice were provided by Dr. Mark Hyer (Medical University of South Carolina). All mice were housed and cared for in the MUSC animal facility and handled according to protocols approved by the MUSC Institutional Animal Care and Use Committee. for lysogeny and isolation of E. Coli strains Gastrointestinal tracts from adult ICR and C57/BL6 mice were collected from fresh cadavers and divided into four segments: (1) stomach and duodenum, (2) 10 cm of small intestine midway between the duodenum and cecum, (3) cecum, and (4) colon and rectum. Intestinal contents were collected by flushing each segment with 1 ml L-Broth containing 5 mM CaCl 2 and 10 mM MgSO 4 , vortexed briefly, and centrifuged at 50 RCF to remove large particulates. Aliquots of supernatant were plated onto MaConkey agar to isolate commensal E. coli. Remaining supernatant was divided and incubated under 5 different conditions to enrich for coliphage: (1) No added host bacteria, no mitomycin C, (2) CN-13 host bacteria and 2 µg mitomycin C/ml, (3) CN-13 host bacteria and no mitomycin C, (4) ER2738 host bacteria and 2 µg mitomycin C/ml, (5) ER2738 host bacteria and no mitomycin C. After 18 h shaking at 37°C, enriched cultures were pelleted by centrifugation at 20,800 RCF. The presence of male-specific phage and somatic phage in the supernatant was assayed by spotting 10 µl onto host cells according to Method 1601 of the United States Environmental Protection Agency for detection of coliphage in fresh water samplesMale specific phage host strain E. coli ER2738 (F'proA + B +
lacI q delta(lacZ)M15zzf::Tn10(Tet R ) and M13mp18 col-
iphage were obtained from New England Biolabs (Beverly
MA). Somatic phage host E. coli strain CN13, and col-
iphages T4 and T7 were purchased from the American
Type Culture Collection, Rockland MD. Coliphage P1 and
PhiX174 were from generously provided by Dr. Caroline
Westwater (Medical University of South Carolina).
Lambda gt11 (Promega, Madison, WI). was generously
provided by Phillip Werner, Medical University of South
Carolina. E. coli bacterial strains grown from de-identified
human clinical specimens were provided by Dr. Lisa
Steed, Dept. of Pathology and Laboratory Medicine,
MUSC. Bacteria were grown in Luria Broth with or with-
out agar (Difco) or on Maconkey agar (Mice
Survey
(page number not for citation purposes)
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AcknowledgementsThis work was supported by a grant from the Medical University of South Carolina University Research Council to LK. Laboratory space and access to equipment were generously provided by James S. Norris, and the MUSC Department of Microbiology and Immunology. Phillip A Werner provided helpful assistance with bacteria typing assays.ELISAELISAs were performed in Reacti-Bind™ maleic anhydride activated clear 96-well plates (Pierce, Rockford IL) coated overnight with one of 6 phages (M13mp18, Lambda, P1, øX174, T4 or T7) in sterile filtered bacterial culture supernatants diluted in PBS. Fecal washes were tested for the presence of phage-specific IgA antibodies, at a dilution of 1:3 in duplicate wells for 2 h at room temperature, using a goat anti-mouse IgA-alkaline phosphatase conjugated secondary antibody (Southern Biotechnology Assoc., Birmingham AL) and Super Signal chemiluminescent substrate (Pierce). Mouse sera were incubated in the same manner except at a 1:6 dilution and utilizing secondary HRP-conjugated antibodies recognizing mouse IgG or IgM (Sigma) to detect phage-specific antibodies. Sera from mice immunized with the laboratory phages by intraperitoneal injection served as positive controls. Horseradish peroxidase conjugated secondary antibodies were detected by incubation with 1-Step ABTS (Pierce).Competing interestsThe author received salary and support from SeptemberAuthors' contributionsLK conceived of and designed the study, carried out the experiments, analyzed the data and prepared the manuscript.
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