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Keywords:

  • Bacteriophage;
  • Campylobacter coli;
  • Campylobacter jejuni;
  • isolation;
  • method;
  • poultry

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  Poultry meat is considered a major source of Campylobacter. This micro-aerobic bacterium is commonly responsible for foodborne illness. This work focuses on the isolation of Campylobacter coli lytic bacteriophages (phages) against target C. coli strains.

Methods and Results:  A method involving the enrichment of free-range chicken samples in a broth containing the target C. coli strains and salts (CaCl2 and MgSO4) was used for phage isolation. This method allowed the isolation of 43 phages that were active against 83% of the C. coli strains used in the isolation procedure. Approximately 65% of the phages were also effective against Campylobacter jejuni strains.

Conclusions:  The use of target pathogens in the phage isolation step improves the likelihood of detecting and isolating phages for the control of these specific strains.

Significance and Impact of the Study:  This technique will be valuable in the context of phage therapy for enriching for phages that are active against specifically identified strains of bacteria, for example from a food poisoning outbreak or epidemic strains resistant to multiple antibiotics. In these situations, using the conventional methods for searching for bacteriophages active for these particular strains can be a time-consuming, if not an unsuccessful process. Using the isolation method described in this manuscript, the particular strains can be added to the enrichment broth increasing the probability of finding phages against them. Therefore, it will shorten the time needed for seeking phages able to lyse target strains, which in most of the cases, because of the rapid increase in antimicrobial-resistant bacteria, is of crucial importance.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Campylobacter is the main cause of enteric illness in the United States and in several member states of the EU (Friedman et al. 2000;Lindqvist et al. 2001; Samuel et al. 2004), and poultry meat products are considered the major source of campylobacteriosis (Jacobs-Reitsma 2000; Shane 2000). Because these organisms are enteric commensals, they are very difficult to control in poultry, and therefore they are present in the derived products, where they are pathogenic to humans (Jacobs-Reitsma 2000). This situation is made worse by recently introduced restrictions on the use of antibiotics in animal production. Phage therapy offers a promising alternative for controlling these pathogens.

Studies of phage therapy for use against Campylobacter have focused mainly on C. jejuni because it is the most commonly isolated Campylobacter species (Tam et al. 2003). Campylobacter coli has been considered the second most frequently isolated Campylobacter species but its importance has been greatly underestimated (Tam et al. 2003). There has been a decrease in C. jejuni and an increase in C. coli recorded in broiler chickens (Avrain et al. 2003; El-Shibiny et al. 2005), and it is recognized that adult birds carry higher numbers of C. coli than young birds (Petersen et al. 2001). The control of C. coli in broilers is therefore of extreme importance. Furthermore, it has been suggested that C. coli is more resistant to some antimicrobials than is C. jejuni (Korolik et al.1998). Aarestrup et al. (1997) demonstrated that the occurrence of resistance for the 16 antimicrobial agents tested was generally high among C. coli isolates compared to C. jejuni isolates.

One of the most important issues in phage therapy is the ability to obtain phages capable of infecting the causal pathogen, hence the isolation step is a critical element. The isolation of Campylobacter phages is considered difficult because of the fastidious growth requirements of the host bacteria. This paper describes an improved method for the isolation of C. coli phages.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Campylobacter strains

The 12 strains of C. coli used in this study were isolated from poultry and poultry products. They were tested for lysogeny (see method below). Three clinical strains of C. jejuni were included in the lytic spectrum study (Table 1). All of the strains were flaA-typed according to the method described by Nachamkin et al. (1993). This is a rapid and cost-effective method (Wassenaar and Newell 2000) that is based on the fact that the flaA gene shows regions of conservation surrounding regions of variability. Therefore, primers can be designed for polymerase chain reaction (PCR), and their products can be used for restriction fragment length polymorphism.

Table 1.   Lytic spectra of the isolated phages against Campylobacter coli and Campylobacter jejuni strains
 C. coli strainsC. jejuni strainsTotal strains infected (%)
FlaA typeABCDEF
Phage hostsPhages*A1189078908891189098910A12A15A3A2A4A18214038208024
  1. Clear lysis areas were marked as (+), turbid lysis areas are represented as (+/−) and absence of lysis was marked as (−).

  2. *Isolated phages phiCcoIBB1-phiCcoIBB43 are represented by the numbers 1–43.

A151+++++/−+/−40
2+++++++++/−+67
5++++27
A36++/−+++/−+++60
7++/−+/−+++/−++++67
8++/−+/−+++/−+++60
9++/−+/−+++/−+++++/−+/−80
10++/−+/−+++/−+++60
890711+++++++++60
12++++/−++−++++++++93
13+++/−+/−++/−+++++/−73
14+++/−+/−++/−+++++73
891023++/−+/−+++/−++++67
24+++/−+/−+++/−++++73
25++/−+/−+++/−+47
26++/−+/−++++/−+53
891127++++++++53
28+++++++++60
29++++++/−+/−47
30++++++/−+47
31++++++40
A432++/−+/−+++/−+++60
A1133++++++++++++/−++93
34++++++++++++/−+87
35++++++++++++++93
36+++++/−33
37++++++++++++++/−93
38+++++++++++73
A239+/−+++27
40+/−++++++47
41+/−++20
42+/−+++27
43+/−+++27

The incubation of all agar plates and broths was at 42°C for 18 h, under micro-aerobic conditions (5% O2, 5% H2, 10% CO2, 80% N2).

Selection of nonlysogenic Campylobacter strains.

Campylobacter strains were screened for lysogeny by using mitomycin C as inducing agent. Briefly, 5 ml of fresh broth growth media (NZCYM broth, N6905; Sigma Aldrich, St Louis, MO) was inoculated with 50 μl of an overnight culture of the strain to be tested for lysogeny. The cultures were incubated at 42°C under micro-aerobic conditions. When they reached the exponential growth phase, mitomycin C was added to a final concentration of 0·5 μg ml−1. The absorbance was measured at 600 nm each hour for 8 h. The cultures were centrifuged (3000 g, 12 min, 4°C), and the supernatant filtered through a membrane filter of 0·45 μm of pore size. A 10-μl aliquot of each of the treated suspensions was spotted onto bacterial lawns prepared individually from each of the 12 strains in NZCYM media (N6905; Sigma Aldrich) supplemented with 0·6% of agar (A6686; Sigma Aldrich) and incubated. The strains that produced a zone of clearing around the spot were omitted from the study. This selection procedure was repeated on three separate occasions.

Isolation of phages

Finely chopped intestines from 12 six-month-old free-range chickens were used for the isolation of phages, according to two different methods. In the first experiment, 10 g of chicken samples was added to SM buffer (0·05 mol l−1 Tris–HCl [pH 7·5], 0·1 mol l−1 NaCl, 0·008 mol l−1 MgSO4) and incubated for 24 h at 4°C. The suspension was centrifuged (8600 g, 10 min), and the aqueous phase was treated with chloroform: one volume per four volumes of supernatant and centrifuged (8600 g, 10 min). It was then used for phage detection (see method below). In the second experiment, the chicken samples (10 g) were mixed with a suspension (50 μl) of each of the 12 C. coli strains [grown individually for 18 h in Bolton Broth (CM0983B; Oxoid, Unipath, Basingstoke, UK) supplemented with selective supplement of antibiotics (Oxoid, SR0208E) and 5% of lysed horse blood (Oxoid, SR0048)] and 10 ml of fresh broth, supplemented with 400 μg ml−1 of CaCl2 and 400 μg ml−1 of MgSO4. This mixture was vortexed, incubated (42°C, micro-aerobic conditions, 18 h) and treated with chloroform as described previously.

Phage detection

A drop of each phage sample (10 μl) was added to lawns of each of the 12 strains of Campylobacter and the plates incubated. Lysis zones were scraped, suspended in 100 μl of SM buffer and replated on a lawn of the Campylobacter strain from which they were originally isolated. Serial dilutions were made to obtain single phage plaques that were propagated three times by this method to ensure purity of the phage.

The isolated phages were named as phiCcoIBB, representing the host C. coli strain (Cco) and the Institute for Biotechnology and Bioengineering (IBB) where the phages were isolated. The phage name is followed by a number given to each particular phage.

Lytic spectra

Each phage was tested against each C. coli and C. jejuni strains (Table 1). Ten microlitres of phage isolate was applied to each bacterial lawn. The plates were incubated, and the appearance of clear zones around the point of application was recorded as the ability to lyse that strain. Three independent experiments of this procedure were performed.

Characterization of phages by Transmission Electron Microscopy (TEM)

Phages were purified by PEG (polyethylene glycol) according to the procedure described by Sambrook and Russell (2001). Phage suspensions were centrifuged at 25 000 g for 60 min and washed twice in ammonium acetate (0·1 mol l−1, pH 7·0). Sediments were deposited on copper grids with carbon-coated Formvar films, stained with 2% (w/v) uranyl acetate (pH 4·2) and studied in a Philips EM 300 electron microscope operating at 60 kV. Magnification was monitored with catalase crystals (Polaron Electron Opticals Ltd, London, UK).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The first phage isolation experiment was performed without the use of an enrichment method. This procedure did not allow the isolation of phages from the chicken samples, using the C. coli strains tested, as no lysis areas were observed (data not shown). Therefore, an alternative methodology was tested in which salts and the target hosts were incubated with the chicken samples (enrichment procedure). All the 12 C. coli strains were used in this enrichment as they were shown to be nonlysogenic. This method allowed the isolation of 43 C. coli phages (data not shown) from six chicken samples. Of the 12 C. coli strains used for phage isolation, only two (A12 and A18) were unable to propagate any of the phages present in the samples.

Of the 43 phages isolated, 10 were discarded because the zones of lysis they produced on their host strain were very turbid. Thus, only the remaining 33 phages were assessed for their lytic spectra (Table 1).

Some of the phages, mainly those isolated using the same C. coli host strain, had similar lytic spectra: phiCcoIBB_6 and phiCcoIBB_8 isolated via C. coli A3, and phiCcoIBB_33 and phiCcoIBB_34 isolated via C. coli A11 (Table 1). Two phages were found to have similar lytic spectra, which had been isolated via different C. coli host strains: phiCcoIBB_14 (host 8907) and phiCcoIBB_24 (host 8910). These two hosts are of the same flaA type (Fig. 1). In general, strains of different flaA types exhibited different susceptibility to the isolated phages. However, some strains with the same flaA type showed different lytic patterns, as did strains A12 and 8910 of flaA type A. Strains A11, A2 and A3 were able to propagate all of the isolated phages. Conversely, C. coli strain A18 was unable to propagate any of the 33 isolated phages. Of the 33 phages, 64% were able to propagate in at least one of the C. jejuni strains tested, and 24% of the isolated phages were able to propagate in all of the C. jejuni tested. Phages phiCcoIBB35 and phiCcoIBB37 which were isolated via host A11 and also phage phiCcoIBB12 isolated via host 8907 showed the broadest lytic spectra, being able to lyse 93% of the Campylobacter strains tested. Therefore, these phages were selected to be characterized by TEM, exhibiting similar structures and sizes, each possessing an icosahedral head and a contractile tail with tail fibers typical of phages from the Myoviridae family (Fig. 2).

image

Figure 1.  Restriction fragment length polymorphism patterns of the flagellin gene flaA digested with DdeI. Lanes are as follows: 1. Ladder 100 bp (Fermentas, GeneRuler 100bp Plus); 2–7. FlaA Type A–F, of the Campylobacter strains used for the lytic spectra; 8- Ladder 100 bp (Fermentas, GeneRuler 100bp Plus).

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image

Figure 2.  Transmission Electron Microscopy images of phages (a) Phage phiCcoIBB35 (b) Phage phiCcoIBB37 (c) phage phiCcoIBB12.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Relatively few researchers have been successful in isolating Campylobacter phages, and the most probable reason for this is inadequate isolation methodology. In the first attempt to isolate Campylobacter phages, we used a methodology previously reported by several authors (Connerton et al. 2004; Atterbury et al. 2005; Loc Carrillo et al. 2005). This method is based on the incubation of chicken samples overnight at 4°C in SM buffer without the addition of Campylobacter strains. As no bacterial hosts were added to the preparation and as the low temperature does not allow phage propagation (Atterbury et al. 2003a), this procedure may have prevented the recovery of phages from the chicken samples in concentrations sufficiently high to ensure the infection in plating stage. The method implemented in our study increased the likelihood of detecting and isolating phage present in very low numbers by using a selective phage-enrichment technique. The samples were incubated with strains of Campylobacter to amplify the phage. It is important to assure that these strains are nonlysogenic because in the case of lysogenic strains, the prophage can escape from the original host and can integrate into a different one (transduction), leading to the transfer of virulence genes and those mediating resistance to antibiotics. Such phages are therefore inappropriate candidates for phage therapy. By selecting just the nonlysogenic strains for the enrichment broth, the probability of isolating this type of phages is avoided.

In the method described, the enrichment broth also contained CaCl2 and MgSO4, as there is evidence that these salts enhance the attachment of the phages to the bacterial hosts (Salama et al. 1989; Aarestrup et al. 2007). Phages are more likely to be found in samples where the levels of Campylobacter colonization are higher, as more hosts are available for phage propagation (Atterbury et al. 2003b). We therefore selected samples likely to contain high numbers of Campylobacter. Free-range chickens are exposed to the outdoor environment, so the likelihood of them becoming colonized by Campylobacter is greater (Hald et al. 2001). The enrichment method developed and used in this study isolated 43 Campylobacter phages from free-range chickens, and these were active against approximately 85% of the C. coli strains used in the isolation step. Some of the phages, namely phage phiCcoIBB12, phiCcoIBB35 and phiCcoIBB37 showed broad lytic spectra and were able to lyse not only the C. coli strains but also the clinical isolates of C. jejuni of different flaA types. These phages are considered good candidates for phage therapy. According to the TEM characterization, they were classified as members of the Myoviridae family, which has been reported in the literature for the majority of Campylobacter phages (Atterbury et al. 2003b; Connerton et al. 2004; Hansen et al. 2007; Loc Carrillo et al. 2007). A detailed physiological and genetic characterization of these phages as well as their in vivo performance to control the levels of Campylobacter-contaminated poultry is being evaluated.

Overall, the method described not only improves the chances of isolating phages by seeding the samples with strains of Campylobacter isolated from chicken, but in so doing isolates phages that can be used subsequently to target those specific strains of Campylobacter. Thus, in the context of phage therapy, a particular pathogen that is causing a flock illness can be isolated using the present method and used to seed chicken samples to isolate specific phages against that particular bacterium. Those phages can then be used for treating the infected flock.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors acknowledge the European Commission under the FP-6-2003-Food-2-A to the project 2005-7224 for the financial support and the Portuguese Science Foundation (FCT) through the grant SFRH/BD/23484/2005.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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