Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland M. Salem1,2, S. Virtanen3, H. Korkeala3 and M. Skurnik1,4 1 Department of Bacteriology and Immunology, Haartman Institute, Research Programs Unit, Immunobiology, University of Helsinki, Helsinki, Finland 2 Department of Microbiology, Faculty of Medicine, University of Benghazi, Benghazi, Libya 3 Department of Food Hygiene and Environmental Health, University of Helsinki, Helsinki, Finland 4 Helsinki University Central Hospital Laboratory Diagnostics, Helsinki, Finland
Keywords bacteriophage, biocontrol, detection, pig, veterinary, Yersinia. Correspondence Mikael Skurnik, Haartman Institute, P.O.Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Helsinki, Finland. E-mail: [email protected] 2014/1636: received 7 August 2014, revised 13 November 2014 and accepted 7 December 2014 doi:10.1111/jam.12722
Abstract Aims: Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. Methods and Results: Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y. enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y. pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y. pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. Conclusions: There was a clear association between the presence of the host bacteria and specific phages in the stools. Significance and Impact of the Study: The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples.
Introduction Bacteriophages (phages) are viruses that infect bacterial cells (Owens et al. 2013), and they carry their genetic information in the form of either DNA or RNA within a protein capsid (Clark and March 2006). Most DNA
phages have tails, by which they bind to specific receptors on the surface of their host bacteria. Phage genome is transferred into the host where it exploits the host RNA, protein and DNA synthesis machineries, to replicate its genetic material and produce new phage particles (Juhala et al. 2000). Phages have been isolated from different
Journal of Applied Microbiology 118, 599--608 © 2014 The Society for Applied Microbiology
599
Yersinia phages in pig stools
M. Salem et al.
sources, such as pigs (Kawano et al. 1983; McLaughlin and King 2008), poultry (Owens et al. 2013), saliva (Bachrach et al. 2003), soil (Salifu et al. 2013), sewage (Kumari et al. 2010) and water (Ul Haq et al. 2012). Bacteria of the genus Yersinia are Gram-negative, facultative anaerobic, nonspore-forming coccobacilli, widely distributed in nature. The genus consists of 17 species, of which Y. enterocolitica, Y. pseudotuberculosis and Y. pestis are pathogenic to humans and animals, and Y. ruckeri is a fish pathogen causing enteric red fish mouth disease. While Y. pestis is the causative agent of plague, Y. enterocolitica and Y. pseudotuberculosis infect the human gastrointestinal tract through contaminated food and/or water, causing a self-limiting gastroenteritis and yersiniosis. In 2010, yersiniosis was reported as the third most common zoonosis in Europe, with a total 6776 infected cases (EFSA 2012). In 2011, the total number of confirmed cases with yersiniosis increased to be 7041 cases (ECDC 2013). Lithuania and Finland showed the highest rates of country-specific notification of yersiniosis (EFSA 2012). In Finland, human yersiniosis is most commonly caused by Y. enterocolitica serotype O:3, and its main reservoir is pigs (Fredriksson-Ahomaa et al. 2006). The consumption of raw or undercooked contaminated pork was the most frequent mode of transmission (ECDC 2013). In Finland, Y. enterocolitica serotype O:5,27 and O:9 and Y. pseudotuberculosis serotype O:1a, O:1b and O:3 strains have also been isolated, but more rarely, from humans (Jalava et al. 2006; Sihvonen et al. 2009). Phage therapy has been suggested as an alternative to treat bacterial infections because of its advantages over antibiotics, including its effectiveness against multidrugresistant bacteria, low cost, and self-replicative mode of action (Wang et al. 2010). Phage therapy should not affect the beneficial bacteria in the body when host specificity is high. The infection of bacteria by lytic (virulent) phages can be detected as clear zones on the bacterial lawn. Lytic phages penetrate into their host cell and multiply using the host’s biosynthetic machinery, and new phage particles accumulate within the cell. Finally, the lysis and death of the host bacteria are caused by phage-encoded lytic enzymes that break the cell wall, and hundreds of infectious phages are released. On the other hand, temperate (lysogenic) phages may enter into a latent state by integrating their genome as part of the host genome or residing as a plasmid and is then called a prophage. This genome insertion can lead to a horizontal transfer of genes, such as those related to toxin production and antibiotic resistance. Lytic phages are preferentially selected to be used for phage therapy (Owens et al. 2013). The aims of this study were as follows: (i) to screen pig stool samples collected from different Finnish pig 600
farms for the presence of phages able to infect selected Yersinia strains, (ii) to determine whether the prevalence of the Yersinia phages in the stool samples correlates positively or negatively with isolation of Yersinia strains from the same samples and (iii) to isolate and characterize the Yersinia-specific phages. Our long-term goal is to test whether the isolated phages could be used to control contamination of pig farms with the pathogenic Yersinia. Materials and methods Isolation of Yersinia from pig stool samples Altogether 793 pig stool samples were collected between November 2010 and March 2012 from 14 different Finnish pig farms. Ten grams of stool samples was thoroughly mixed in Erlenmeyer flasks with 90 ml of PMB broth (phosphate-buffered saline with 05% peptone, 1% mannitol and 015% bile salts), and after particulate material had settled down, 100 ll of the supernatant was immediately plated onto cefsulodin-irgasan-novobiocin (CIN) agar (Oxoid, Cambridge, UK). Additional 7-day cold enrichment and 14-day cold enrichment of the PMB suspension at 4°C were used, followed by 100 ll plating onto CIN agar (Laukkanen et al. 2010). With the 14-day cold enrichment, a 05 ml volume of PMB culture was transferred into 45 ml 025% potassium hydroxide (KOH) in 075% NaCl for 20 s before plating. KOH has been used to make the isolation process more selective for Yersinia (Laukkanen et al. 2010). CIN agars were incubated at 28°C for 24 h and further 24 h at room temperature. Typical small (
ORIGINAL ARTICLE
Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland M. Salem1,2, S. Virtanen3, H. Korkeala3 and M. Skurnik1,4 1 Department of Bacteriology and Immunology, Haartman Institute, Research Programs Unit, Immunobiology, University of Helsinki, Helsinki, Finland 2 Department of Microbiology, Faculty of Medicine, University of Benghazi, Benghazi, Libya 3 Department of Food Hygiene and Environmental Health, University of Helsinki, Helsinki, Finland 4 Helsinki University Central Hospital Laboratory Diagnostics, Helsinki, Finland
Keywords bacteriophage, biocontrol, detection, pig, veterinary, Yersinia. Correspondence Mikael Skurnik, Haartman Institute, P.O.Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Helsinki, Finland. E-mail: [email protected] 2014/1636: received 7 August 2014, revised 13 November 2014 and accepted 7 December 2014 doi:10.1111/jam.12722
Abstract Aims: Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. Methods and Results: Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y. enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y. pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y. pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. Conclusions: There was a clear association between the presence of the host bacteria and specific phages in the stools. Significance and Impact of the Study: The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples.
Introduction Bacteriophages (phages) are viruses that infect bacterial cells (Owens et al. 2013), and they carry their genetic information in the form of either DNA or RNA within a protein capsid (Clark and March 2006). Most DNA
phages have tails, by which they bind to specific receptors on the surface of their host bacteria. Phage genome is transferred into the host where it exploits the host RNA, protein and DNA synthesis machineries, to replicate its genetic material and produce new phage particles (Juhala et al. 2000). Phages have been isolated from different
Journal of Applied Microbiology 118, 599--608 © 2014 The Society for Applied Microbiology
599
Yersinia phages in pig stools
M. Salem et al.
sources, such as pigs (Kawano et al. 1983; McLaughlin and King 2008), poultry (Owens et al. 2013), saliva (Bachrach et al. 2003), soil (Salifu et al. 2013), sewage (Kumari et al. 2010) and water (Ul Haq et al. 2012). Bacteria of the genus Yersinia are Gram-negative, facultative anaerobic, nonspore-forming coccobacilli, widely distributed in nature. The genus consists of 17 species, of which Y. enterocolitica, Y. pseudotuberculosis and Y. pestis are pathogenic to humans and animals, and Y. ruckeri is a fish pathogen causing enteric red fish mouth disease. While Y. pestis is the causative agent of plague, Y. enterocolitica and Y. pseudotuberculosis infect the human gastrointestinal tract through contaminated food and/or water, causing a self-limiting gastroenteritis and yersiniosis. In 2010, yersiniosis was reported as the third most common zoonosis in Europe, with a total 6776 infected cases (EFSA 2012). In 2011, the total number of confirmed cases with yersiniosis increased to be 7041 cases (ECDC 2013). Lithuania and Finland showed the highest rates of country-specific notification of yersiniosis (EFSA 2012). In Finland, human yersiniosis is most commonly caused by Y. enterocolitica serotype O:3, and its main reservoir is pigs (Fredriksson-Ahomaa et al. 2006). The consumption of raw or undercooked contaminated pork was the most frequent mode of transmission (ECDC 2013). In Finland, Y. enterocolitica serotype O:5,27 and O:9 and Y. pseudotuberculosis serotype O:1a, O:1b and O:3 strains have also been isolated, but more rarely, from humans (Jalava et al. 2006; Sihvonen et al. 2009). Phage therapy has been suggested as an alternative to treat bacterial infections because of its advantages over antibiotics, including its effectiveness against multidrugresistant bacteria, low cost, and self-replicative mode of action (Wang et al. 2010). Phage therapy should not affect the beneficial bacteria in the body when host specificity is high. The infection of bacteria by lytic (virulent) phages can be detected as clear zones on the bacterial lawn. Lytic phages penetrate into their host cell and multiply using the host’s biosynthetic machinery, and new phage particles accumulate within the cell. Finally, the lysis and death of the host bacteria are caused by phage-encoded lytic enzymes that break the cell wall, and hundreds of infectious phages are released. On the other hand, temperate (lysogenic) phages may enter into a latent state by integrating their genome as part of the host genome or residing as a plasmid and is then called a prophage. This genome insertion can lead to a horizontal transfer of genes, such as those related to toxin production and antibiotic resistance. Lytic phages are preferentially selected to be used for phage therapy (Owens et al. 2013). The aims of this study were as follows: (i) to screen pig stool samples collected from different Finnish pig 600
farms for the presence of phages able to infect selected Yersinia strains, (ii) to determine whether the prevalence of the Yersinia phages in the stool samples correlates positively or negatively with isolation of Yersinia strains from the same samples and (iii) to isolate and characterize the Yersinia-specific phages. Our long-term goal is to test whether the isolated phages could be used to control contamination of pig farms with the pathogenic Yersinia. Materials and methods Isolation of Yersinia from pig stool samples Altogether 793 pig stool samples were collected between November 2010 and March 2012 from 14 different Finnish pig farms. Ten grams of stool samples was thoroughly mixed in Erlenmeyer flasks with 90 ml of PMB broth (phosphate-buffered saline with 05% peptone, 1% mannitol and 015% bile salts), and after particulate material had settled down, 100 ll of the supernatant was immediately plated onto cefsulodin-irgasan-novobiocin (CIN) agar (Oxoid, Cambridge, UK). Additional 7-day cold enrichment and 14-day cold enrichment of the PMB suspension at 4°C were used, followed by 100 ll plating onto CIN agar (Laukkanen et al. 2010). With the 14-day cold enrichment, a 05 ml volume of PMB culture was transferred into 45 ml 025% potassium hydroxide (KOH) in 075% NaCl for 20 s before plating. KOH has been used to make the isolation process more selective for Yersinia (Laukkanen et al. 2010). CIN agars were incubated at 28°C for 24 h and further 24 h at room temperature. Typical small (
