Isolation and characterization of bacteriophages of Helicobacter pylori ...

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Research Article

Isolation and characterization of bacteriophages of Helicobacter pylori isolated from Egypt Mahmoud EF Abdel-Haliem*1 & Ahmed Askora1 Department of Botany, Faculty of Science, Zagazig University, 44519, Zagazig, Egypt *Author for correspondence: Tel.: +20 111 459 8085 n Fax: +25 5230 8213 n [email protected] 1

Aim: The aim of this work was to isolate and characterize bacteriophages from wastewater for the pathogenic bacteria Helicobacter pylori. Materials & methods: In this study, we isolated and characterized two phages against H. pylori isolated from gastric biopsies. The specific phages were isolated by the single plaque isolation technique, propagated by the liquid enrichment method and purified by the polyethylene glycol–dextran sulfate two-phase system. Results: The phages were designated as fHPE1 and fHPE2; fHPE1 had a head with a diameter of approximately 62 nm and short noncontractile tail with a length and width of approximately 12 × 6 nm and fHPE2 had an isometric head with a diameter of approximately 92.5 nm and a tail with a length of approximately 180 nm and width of approximately 15 nm. The host ranges were investigated with four strains of Helicobacter and all strains were susceptible to either fHPE1 or fHPE2. Conclusion: These results suggest that it may be possible to use this phage to control the disease caused by H. pylori.

Helicobacter pylori is a Gram-negative, microaerophilic, pathogenic bacterium that can efficiently colonize the gastric epithelial cells of primates and is an etiologic agent of chronic gastritis [1,2]. Chronic, persistent infection by H. pylori may cause many gastric diseases, including chronic atrophic gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma and gastric cancer [3–5]. It is estimated that H. pylori infects at least half of the world’s population [2]. Treatment of H. pylori gastritis with appropriate antibiotics will usually result in the rapid elimination of these organisms; however, the emergence of antibiotic resistance often decreases the eradication rates of H. pylori infections. Many factors have been implicated as causes of treatment failure, but the main antibiotic resistance mechanisms described to date are due to point mutations in the bacterial chromosome, a consequence of significant phenotypic variation in H. pylori [6]. Hence, the prevalence of antimicrobial resistance in H. pylori increases the need to search for new therapeutic strategies and alternative anti microbials effective against H. pylori infection [6]. Several alternative treatments against H. pylori not involving the use of antibiotics are under investigation. The use of bacteriophages to treat infectious diseases has been considered. Bacteriophage therapy consists of the use of bacteriophages that specifically lyse target bacteria, and was developed and applied in the preantibiotic era. Very little is known about H. pylori phages; however, shortly after the discovery of H. pylori, 10.2217/FVL.13.58 © 2013 Future Medicine Ltd

intracellular phage-like particles were described in human gastric mucosa [7,8]. Two other studies from the early 1990s showed spontaneous production of small amounts of phage particles by the H. pylori strain [9]. Furthermore, Vale et al. described H. pylori temperate induction of phage‑like particles using UV light [10]. The ongoing sequencing of bacteriophage genomes has produced unprecedented amounts of data, which is needed for understanding phage biology. Very recently, two complete genome sequences of H. pylori phages KHP30 and KHP40 were isolated from the culture supernatants of H. pylori (east Asian-type isolates) from Japanese patients [11]. Furthermore, Uchiyama et al. have characterized a novel spherical H. pylori phage KHP30, and this phage was stable over a wide pH range [12]. The genome sequence of these phages will extend our understanding of the process of coevolution of H. pylori and its phages. The aim of this study was to isolate and characterize bacteriophages that specifically infect H. pylori and investigate their effectiveness against pathogenic strains of H. pylori in order to be able to select effective phages for future biocontrol. Materials & methods Isolation of H. pylori & culture conditions

Gastric biopsy specimens were obtained from patients in Zagazig, Egypt. Each biopsy specimen was spread onto blood agar, supplemented with trimethoprim, vancomycin, amphotericin B and polymyxin B, and plates were incubated in Future Virol. (2013) 8(8), 821–826

Keywords n

H. pylori n phages n plaques Siphoviridae

n Podoviridae n

part of

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Abdel-Haliem & Askora

microaerophilic conditions. Bacterial isolates consistent with H. pylori in shape, colony morphology, enzymatic activity and Gram-negative status grew within 7–10 days. Single-colony isolates were subcultured on blood agar plates to isolate genomic DNA. Identification of H. pylori strain by sequencing 16S rRNA

The isolated H. pylori were confirmed by sequencing 16S rRNA. Total DNA was extracted from eight isolated colonies of bacteria [13]. The gene coding for 16S rRNA was amplified from each isolate by PCR with universal primers (forward primer [F27] 5´-AGAGTTTGATCCTGGCTCAG-3´ [14] and reverse primer [R1492] 5´-GGTTACCTTGTTACGACTT-3´ [15]). These primers bind to universally conserved regions and permit the amplification of an approximately 1500‑bp fragment. The PCR amplification was carried out in a GeneAmp® PCR system 9600 thermocycler (Perkin Elmer, ON, Canada). The amplification conditions were as follows: 94°C for 10 min and 35 cycles of denaturation at 95°C for 30 s; annealing–extension at 56°C for 1 min and 72°C for 1 min; and an extension at 72°C for 10 min. The presence and yield of specific PCR products (16S rRNA) were monitored by running 1% agarose gels. Then, the PCR product was cleaned up using a GeneJET™ PCR Purification Kit (Thermo Fisher Scientific, MA, USA). Amplified DNA fragments were partially sequenced at GATC Biotech AG (Germany) with an 3730xl DNA sequencer (Applied Biosystems, CA, USA) using the forward primer (F27). Sequence ana lysis and comparison to published sequences was made using the Basic Local Alignment Search Tool (BLAST) program [16,101]. Nucleotide sequence accession number

The sequence data for the 16S rRNA of H. pylori have been deposited in the GenBank database under accession number JX455160. Isolation of H. pylori phages

H. pylori phages were isolated from wastewater obtained from Zagazig. A total of 50 ml of wastewater was filtered through a 0.45‑µm membrane filter and mixed with 50 ml of LB broth) containing the log-phase cells of H. pylori. After 48 h of growth at 37°C, the culture was centrifuged and filtered through a 0.45‑µm membrane filter. The presence of lytic phages in the filtrate was examined by using the double-layer method with some modifications [13]. A total of 100 µl of the filtrate was mixed with 400 µl of log-phase culture 822

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of H. pylori and incubated at 37°C for 30 min. The mixture was added to 3.5 ml of molten top agar (0.7% agar), which was already cooled down to 50°C, mixed gently and poured into agar plate. The plate was left to stand at room temperature for 30 min to allow the top agar to solidify. The presence of lytic phages in the form of plaques was detected after incubation of the plate at 37°C for 48 h. Propagation & purification of H. pylori phages

A single plaque was extracted with a sterile glass Pasteur pipette and put into a log-phase culture of H. pylori. After incubation at 37°C for 48 h, the phage–host mixture was centrifuged at 15,000 rpm for 10 min and filtered through a 0.45‑µm membrane filter. The filtrate was subjected to the double-layer method as mentioned above. Three repeated rounds of single-plaque isolation and reinoculation were performed. The phage was eluted from the final resulting plate by adding 5 ml of saline solution (0.85% NaCl) on top of the plate and being incubated at 4°C overnight with shaking. The phage-containing buffer retrieved from the plate was centrifuged at 15,000 rpm for 10 min and filtered through a 0.45‑µm membrane filter. The two isolated phages were propagated by the liquid culture method. Phages were added to the main sensitive strain (H. pylori Zag1) in 1000‑ml Erlenmeyer flasks with a ratio of 1:10 (v/v) and incubated at 37°C for 3 days. The propagated phages (1200 ml of each) were purified by a dextran sulfate-polyethylene glycol two-phase system. [17]. Weights of 222.3, 0.48, 15.8 and 4.2 g of phage lysate, dextran sulfate 500, polyethylene glycol (PEG 6000) and NaCl, respectively, were mixed in a separating funnel to give a mixture containing a ratio of 5, 0.2 and 1.7% (w/w) of PEG 6000, dextran sulfate 500 and NaCl, respectively. After mixing, the funnel was allowed to stand at 4°C overnight. A heavily turbid bottom layer was slowly collected into a clear tube and centrifuged at 2000 rpm for 10 min, the clear top and bottom phases were removed by pipette and the remaining interface ‘cake’ was suspended in 2.5 ml of 0.1% (w/w) dextran sulfate solution, then 0.15 ml of a 3 M KCl solution was added to each milliliter of suspension. The mixture was allowed to stand for 24 h at 4°C and centrifuged at 2000 rpm for 10 min. After centrifugation, the supernatant containing phages was obtained and dialyzed against saline solution (0.85% NaCl) at 4°C for 72 h. After dialysis, the phage suspensions were centrifuged at 15,000 rpm for 2 h at 4°C, then the supernatants were discarded, and then future science group

Isolation & characterization of bacteriophages of Helicobacter pylori from Egypt

Research Article

100 nm

100 nm

Figure 1. Electron micrographs of the negatively stained isolated Helicobacter pylori phages. (A) Transmission electron micrograph of Helicobacter pylori phage fHPE1. (B) Transmission electron micrograph of H. pylori phage fHPE2. Tail structures of fHPE1 are indicated with an arrow.

the pellets were resuspended in 2.3 ml of saline solution (0.85% NaCl) and assayed. Determination of phage titer

The phage-containing solution was serially diluted in distilled water. Each dilution was subjected to plaque assay using the double layer method as mentioned earlier. Plaques were counted in the plates containing 50–300 plaques and expressed as PFU/ml.

slight modifications: 100 µl of a log-phase culture of each tested bacteria were added to 3.5 ml of 50°C molten soft agar (0.7% agar), mixed gently and poured into an agar plate. After solidification, 10 µl of the phage suspension was spotted on the lawn of bacteria. After the plate was left to stand for 30 min at room temperature, it was incubated at an appropriate temperature for 48 h before checking for the presence of a clear zone on the plate, which indicated the ability of the phage to infect the tested bacteria.

Electron microscopy

The isolated phages were examined as described previously [18]. A drop of each phage suspension (107 PFU/ml) was placed on 200‑mesh copper grids with carbon-coat Formvar® (Sigma-Aldrich, MO, USA) films and excess was drawn off with filter paper. A saturated solution of uranyl acetate was then placed on the grids and excess was drawn off as before. Specimens were examined with an electron microscope (model: JEM‑1010) at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Physical properties of the isolated phages

pH stability and thermal stability tests were carried out, and equal amounts of phage particles were treated under the specified conditions. Samples were taken at different time intervals and supernatants from centrifugation were used directly in the assays.

Results Identification of H. pylori clinical strains

From the gastric biopsy that was collected from the patients, four different strains of H. pylori were isolated. The four isolates (H. pylori Zag1, H. pylori Zag2, H. pylori Zag3 and H. pylori Zag4) were tested for their susceptibility to phage infection by using them individually as indicators. H. pylori Zag1 was the strain that was most sensitive to the isolated phages. The identification of this strain was confirmed by using sequence information derived from their 16S rRNA. Table 1. Effect of pH on the infectivity of isolated phages. fHPE1

pH

fHPE2

PFU/ml × 107

%

PFU/ml × 107

%

4

0

0

0

0

5

3.64

49.1

3.55

51.4

6

4.53

61.2

4.99

72.3

Host range

7 (control)

7.40

100

6.90

100

In order to determine their host range, phages fHPE1 and fHPE2 were tested against four bacterial strains. Bacteriophage lysis assays were conducted based on the double-layer method with

8

2.35

31.7

2.13

30.8

9

1.17

15.8

0.89

12.8

10

0

0

0

0

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Table 2. Effect of temperature on the infectivity of isolated phages. fHPE1

Temperature (°C)

fHPE2

PFU/ml × 107

%

PFU/ml × 107

%

37 (control)

7.40

100

6.90

100

40

7.11

96.7

6.11

88.5

45

6.36

85.9

5.26

76.2

50

5.20

70.2

4.22

61.1

55

3.33

45.0

2.54

36.8

60

1.49

20.1

1.37

19.8

65

0.26

3.5

0.34

4.9

70

0

0

0

0

Isolation & purification of H. pylori bacteriophages

The presence of bacteriophages specific for H. pylori strains were detected using the spot test technique. Phage suspensions were obtained and assayed quantitatively by the double-layer method. Phage plaques were detected at frequencies of 5–40 plaques per plate from different samples (all from Zagazig City) and were further tested for their host specificity. These plaques were isolated and propagated in strain H. pylori Zag1 and were assayed qualitatively by the double-layer method. Single plaques resulting from the high dilutions of phages (10 -6 PFU/ml) were selected and extracted based on their morphology (size and shape; Figure 1). Two plaques with different diameters were chosen and designated as fHPE1 and fHPE2. Each plaque was added to 3 ml of liquid culture of H. pylori (108 CFU/ml) and incubated at 37°C for 48 h; then, phage lysates were prepared and assayed quantitatively. Phages were assayed quantitatively after concentration, and their titers were approximately 109 and 1010 PFU/ml for fHPE1 and fHPE2, respectively. Morphology of H. pylori phages

Purified, concentrated phages specific for H. pylori (fHPE1 and fHPE2) were negatively stained with uranyl acetate and examined by transmission electron microscopy in order Table 3. Host range specificity of the isolated phages. Strain

fHPE1

fHPE1

Helicobacter pylori Zag1

+

+


+

-


+

+


-

-

A positive indication (+) means that the strain is susceptible to the phage, while a negative indication (-) means that no plaques were observed.

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to determine the morphotype of the phages. According to the transmission electron micrographs in Figure 1, phage fHPE1 belongs to the Podoviridae family, which is characterized by phages with a short noncontractile tail. fHPE1 has a very short tail, most likely being type C in Bradley’s classification [19]. The diameter of the head is approximately 62 nm, and it has a short noncontractile tail with a length and width of approximately 12 × 6 nm. The fHPE2 belongs to the Siphoviridae family and has an isometric head with a diameter of approximately 92.5 nm and a tail with a length of approximately 180 nm and a width of approximately 15 nm (Figure 1). Physical properties of the isolated phages

The optimal pH was determined by testing the stability of phages fHPE1 and fHPE2 under different pHs. Almost no reductions of infectious phage fHPE1 and fHPE2 were observed at pH 7.0, while different reduction percentages were obtained at other pHs, with 49.1 and 51.4% recovery of infectious phages fHPE1 and fHPE2 at pH 5, respectively (Ta ble 1). These results suggest that both phages were stable from pH 5 to 9, suggesting that it is adapted to the acidic environment of the human stomach. A thermal stability test was carried out to analyze the heat-resistant capability of fHPE1 and fHPE2 phages at pH 7.0. The preliminary experiments showed that fHPE1 and fHPE2 stock solutions retained almost 100% infection activity after incubation at 37°C for 1 month (data not shown), so higher temperatures of 40, 45, 50, 55, 60, 65 and 70°C were chosen to test the thermal inactivation point of fHPE1 and fHPE2 (Table 2) as a feature of phage characterization. The results showed both phages fHPE1 and fHPE2 were extremely heat-stable, remaining alive after 60 min incubation at 65°C, while more than 99% of phages lost their infection ability after 15 min at 70°C. Determination of phage host range

The lytic activity of isolated phages was examined in H. pylori bacterial strains (Table 3). In this experiment, the host ranges of fHPE1 and fHPE2 phages specific for H. pylori were determined using four strains of H. pylori (Table 3). The host range of fHPE1 is incompatible with that of fHPE2, in that fHPE1-susceptible strains are resistant to fHPE1 and vice versa, except for strains H. pylori Zag1 and H. pylori Zag3, which are susceptible to both phages (Table 3). future science group

Isolation & characterization of bacteriophages of Helicobacter pylori from Egypt

Discussion & conclusion

Bacteriophages constitute the majority of biological organisms on Earth and have crucial influences on the evolution of bacteria [20–23]. With an estimated 1031 bacteriophages [24], an appreciation of the diversity and complexity of these organisms can only be gauged by the current body of knowledge produced by their study. As new phages are discovered, our understanding of molecular biology and microbial genetics will continue to grow and new technologies will be developed that will continue to help molecular, cellular and developmental biologists understand the complexity of life [25]. In this study, we isolated and characterized two bacteriophages specific for H. pylori. The phages were designated fHPE1 and fHPE2. Morphologically, fHPE1 had a head with a diameter of approximately 62 nm and a short noncontractile tail with a length and width of approximately 12 × 6 nm, and fHPE2 had an isometric head with a diameter of approximately 92.5 nm and a tail with a length of approximately 180 nm and a width of approximately 15 nm. Their host ranges were investigated with four strains of H. pylori and all strains were susceptible to either fHPE1 or fHPE2. Furthermore, the genomic DNA of the isolated phages were extracted and the DNA was analyzed by electrophoresis at 100 V in a 1.0% agarose gel stained with ethidium bromide, and both phages were shown to have dsDNA (data not shown). The DNA of the isolated phages is now being analyzed using gel pulsed electro phoresis to determine the approximate genome size of each phage. Since potential applications of candidate phage isolates as agents of pathogen control would likely be subjected to a holding period prior to distribution and application, the physical properties of the phage particles may become parameters of relative importance in phage therapy applications. Interestingly, both phages were stable from pH 5 to 10, suggesting that they are adapted to the acidic environment

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of the human stomach. Thermal stability tests were carried out to analyze the heat-resistant capability of fHPE1 and fHPE2 at pH 7. The results showed both phages fHPE1 and fHPE2 were extremely heat-stable, remaining infective after 60 min incubation at 65°C, while more than 99% phages lost their infection ability in 15 min at 70°C. Despite being an evolution in treatment, there is an increasing percentage of failure of antibiotic therapy due to antibiotics resistance. Phage therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections, and H. pylori is a good target. However, there are no available phage collections for this organism, and H. pylori phage description is rare in the literature. Now that the isolated phages in this study have been successfully characterized, the next step in assessing them for potential therapeutic utility will be to examine them for virulence factors and/or tendencies towards horizontal gene transfer. The digestion with restriction enzymes and sequencing of phage DNA will be a priority in future studies. Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary The prevalence of antimicrobial resistance in Helicobacter pylori increases the need to search for new therapeutic strategies, and bacteriophages have recently been evaluated as potential treatments for a number of multidrug-resistant bacteria, such as Pseudomonas aeruginosa, among others. Bacteriophages could be an important alternative to antibiotics, especially for the treatment of multidrug-resistant bacteria. In this study, two phages against H. pylori were isolated from gastric biopsies, characterized and designated as fHPE1 and fHPE2. Transmission electron micrographs show that the fHPE1 phage belongs to the Podoviridae family, while fHPE2 belongs to Siphoviridae. Several lines of experiments showed all H. pylori strains used in this study are susceptible to either fHPE1 or fHPE2. Both phages were extremely heat-stable and were adapted to the acidic environment of the human stomach. These results suggest that it may be possible to use this phage to control disease caused by H. pylori; however, more phages should be isolated to allow for the preparation of ‘phage cocktails’, which might be a better method for the application of phages in this situation.


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