Diversity of Phage-Host Specificity in <em>Brucella</em> Phage

Special Article – Animal Brucellosis

J Bacteriol Mycol. 2017; 4(2): 1049.

Diversity of Phage-Host Specificity in Brucella Phage

Antadze I¹, Dadunashvili M¹, Burbutashvili T¹, Gunia S¹, Balarjishvili N¹, Tevdoradze E¹, Pataridze T¹, Obiso RJ², Hagius S³, Elzer P³ and Kutateladze M¹*

¹George Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, Georgia

²Avila Scientific, LLC, Christiansburg, Virginia, USA

³Louisiana Agricultural Experiment Station, Baton Rouge, Louisiana, USA

*Corresponding author: Mzia Kutateladze, George Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, Georgia

Received: March 24, 2017; Accepted: April 27, 2017; Published: May 04, 2017


Bacteriophage typing of Brucella is accepted as an additional tool that can be used for the identification of bacterial species by the World Health Organization’s Expert Committee of Brucellosis. Phage typing is based on the host specificity of bacteriophages. Few phage for bacterial species are currently validated for typing purposes. Tbilisi (Tb) is a unique phage isolated in the 1950s at the Eliava Institute of Bacteriophages, Microbiology, and Virology in Tbilisi, Georgia. This paper describes some historical studies of Brucella phage performed at the Eliava Institute, as well as a comparative characterization of several novel Brucella bacteriophages from the Institute’s collection. Phage– host specificity was also examined using DNA restriction and is demonstrated by the efficient plating of bacteriophages grown on different Brucella hosts. The lytic reactions of the Brucella phage used in this study confirm the data that was also obtained by serological and molecular genotyping methods. Studies of the mechanisms of phage-host specificity support the use of phage typing schemes for the identification of bacterial strains of Brucella.

Keywords: Bacteriophage; Host-specificity; Efficiency of plating


Brucellosis – a zoonotic disease and potential biological weapon - is caused by the bacterial genus of Brucella. The bacteria are transmitted from animals to humans by ingestion of infected food products, direct contact with an infected animal, or inhalation of aerosols. This disease continues to be a major public health concern worldwide and is a very common zoonotic infection throughout the world. Definitive diagnosis of this disease is based on culture (identification of the pathogen), serology, and molecular biological methods. Serological methods traditionally have been used to speciate Brucella isolates. Serotyping results should ideally be confirmed by molecular genotyping, a variety of which are available for this purpose: polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [1]; cytoplasmic protein-specific gene probe analysis [2]; Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) [3,4]; or typing with the rpoB gene coding the DNA-dependent RNA polymerase (RNAP) ß subunit [5,6]. In addition to these molecular methods, phage typing is used as an additional confirmatory tool for the identification of Brucella species.

Phage typing using species-specific bacteriophages has been used to differentiate Brucella species for many years [7]; the phage Tb, Iz, Wb, Bk, and S708 are those most often used for typing purposes [7]. Despite their utility, the biology of these bacterial viruses is not well understood, particularly with respect to their interactions with host bacterial cells. Brucella phage were first isolated at the Tbilisi Institute of Vaccines and Sera (the prior name of the Eliava Institute of Bacteriophages, Microbiology, and Virology) in Georgia by Nemsadze, Popkhadze, and Kilasonidze in 1952 [8] and used to evaluate the activity of phage filtrates on museum cultures and freshly isolated cultures of Brucella on solid media.

Improved methods for the isolation and target-specific reinforcement of phage, together with parallel research on the antigenic structure of Brucella, led to the isolation of a considerable number of phage from various sources from the 1950s through the 1970s [9-14]. Twenty-three Brucella bacteriophages, which were stable at high concentrations [titer 10-4 – 10 by the method developed by Appelmans [15]] were isolated between 1955 and 1962 at the Eliava Institute. Seven of these phage were isolated from the environment, 15 from the blood of human brucellosis patients, and one from a person vaccinated with a live Brucella vaccine [16,17]. All 23 phage were specific to B. abortus, despite the detection of B. melitensis in the blood of three of the brucellosis patients from whom the phage were isolated [16].

Brucella phage typing has a long history [18-23]. The stable Brucella phage named Tb, was first isolated from manure in 1955 at the Tbilisi Institute of Vaccines and Sera [23]. Subsequently, this phage was approved by the International Subcommittee on the Taxonomy of Brucella as a reference phage for the diagnostics and differentiation of Brucella strains [25]. Tb phage have been studied and used for typing purposes by many scientists [25-28]. One series of experiments showed that Tb phage are specific to B. abortus; plaques were not observed on lawns of B. suis, although phage at high concentrations (104 x routine test dilution or greater) did cause inhibition of growth that resembled lysis. High concentrations of Tb phage have also been shown to inhibit the growth of B. melitensis [29].

Phage typing has been used to confirm Brucella species. In two previous studies, 543 Brucella strains from different countries (former Soviet Union, United Kingdom, Poland, Germany, and South Africa) were identified [23,29]. Preliminary speciation was carried out using standard bacteriological and biochemical tests. The results of these studies indicated that 277 strains were B. melitensis, 62 strains were B. abortus, and 204 strains were B. suis. The results of the phage susceptibility testing demonstrated that 177 of the 277 B. melitensis strains in the Eliava Institute collection belonged to a single group according to the phage typing scheme described by Morgan [25]; a total of 99 non-Eliava strains also belonged to the same group. Seventy-three strains that were lysed by both dilutions of phage were identified as the B. abortus biotype V. All but five B. abortus strains were lysed by Tb phage; further investigation indicated that these five strains already contained temperate phage and were not lysed. Among the B. suis strains, 40 out of 204 were lysed by phage.

Currently, the Eliava Institute collection includes 44 Brucellaspecific bacteriophages, including the Tb phage; one phage isolated from B. canis; four phage isolated from B. ovis; 19 phage isolated from B. melitensis; 17 phage isolated from B. abortus; and two phage isolated from B. suis. All of the phage in the collection are specific; the phage isolated from B. abortus and B. melitensis cause lysis only in B. abortus and a limited number of B. melitensis strains. Phages isolated from B. ovis and B. canis fully lyse B. abortus and partially lyse B. melitensis but do not lyse B. ovis, B. suis, or B. canis. The current paper describes features of a set of 10 bacteriophages that have been selected for typing purposes for various Brucella species. The bacteriophages outlined in this paper were characterized, including an analysis of their reproduction parameters and lytic specificity against various species of Brucella.

Materials and Methods

Some of the bacterial strains of Brucella used in the study were from the Eliava Institute’s bacterial collection: B. abortus S19 vaccine strain; B. abortus 141, serotype I, originally isolated in Russia; B. abortus 544 serotype I; B. abortus 99 serotype V, was originally from the UK Weybridge collection; and B. abortus 64 serotype III, was isolated in Tbilisi. Among the B. melitensis strains in the Eliava museum, N7 was obtained from Saratov, Russia in 1963; and N16 was isolated in Moscow in 1962. The strain N110 was isolated from human synovial fluid in Tbilisi in 1942; N 237 and 238 were isolated from the blood of a brucellosis patient in Tbilisi in 1959. The strains N 71 m/z and 70 v/z were originally isolated from a patient in Bulgaria; 130 m/z was isolated from a patient in Germany; and 238 m/z and 254 m/z were originally isolated from a cow in England. Strain 63/9 was received from Almaty, Kazakhstan in 1979. Additional bacteriophages typing was conducted using bacterial strains that reside at the Louisiana State University (LSU) Ag Center and were as follows: B. inopinata BO1 from a breast cancer implant in a brucellosis patient [30]; B. inopinata BO2 from the lung biopsy of a patient with chronic pulmonary destructive pneumonia [31]; Brucella strain NF2653 from wild native rodents in Australia [32]; and SDRL an atypical B. abortus strain from a rat liver sample from San Diego, California [33]. The following Brucella phage were used for this study: Tb, originally isolated from manure; phage 1066 from B. canis; 281; 02 from B. ovis; 177; 110 from B. melitensis; V; 544; 141 from B. abortus; and 11sa from B. suis. All bacteriophages were propagated on two bacterial strains: B. abortus S19 and B. abortus 141 strains.

Phage spot test

The phage spot test is a common tool to determine phage host specificity. The host range of each phage (phage specificity) was determined by spotting 10 µL of a phage suspension (~109 PFU/mL) in nutrient broth onto freshly prepared bacterial lawns and counting the plaques that appeared after 24 and 48 hours of incubation at 37°C. Visual characteristics of the phage plaques (PFU/mL) were also evaluated. The Routine Test Dilution (RTD) is defined as the highest dilution of the phage stock that will produce confluent lysis of a lawn inoculum of the propagating strain. The results of the phage spot test were determined from three sets of experiments after each of the 24 hour and 48 hour time points.

Electron microscopy

The morphology of the phage particles was studied using an electron microscope JEM x100 (JEOL). Parlodion plates were overlaid with 1010 PFU/mL phage suspensions with uranyl acetate as a contrast agent.

Phage structural proteins

Phage suspensions (60 µL at 1010 to 1011 PFU/mL) were added to 13 µL of sample buffer containing 8% sodium dodecyl sulfate, 0.1% glycerol, and 0.5 % bromophenol blue; 5.0% ß-mercaptoethanol was added to the solution prior to use. Samples were boiled for ten minutes and then loaded onto 10% polyacrylamide gels [34]. Electrophoresis was carried out at 60 V, 9 mA for 18 hours in tris-glycine buffer.

Phage biology study

Biological properties, mainly phage-host interaction parameters including adsorption, latent period, lysis time, and average burst size, were calculated by standard methodology [35].

DNA isolation and restriction

Phage DNA was isolated by standard phenol/chloroform deproteinization [36] and with QIAamp DNA mini kits (Qiagen). Several restriction endonucleases were used according to manufacturer’s instructions (Biolab). Enzyme-restricted DNA fragments were subjected to electrophoresis on agarose gels. The gels were photographed with ultraviolet illumination.

Results and Discussion

For phage typing, ten bacteriophages (Tb, 141, 281, 544, 1066, 11sa, 02, 177, V, and 110) from the Eliava collection were selected based on the specificity of their lytic reaction on different Brucella species.

Morphology of phage

The size and shape of phage plaques varies considerably among Brucella phage [37-39]. In general, plaques range from 0.2 mm to 4 mm and are polymorphic on bacterial lawns; some of them are oval or perfectly round while others are more irregular. All Brucella phage from the Georgian collection, including the Tb phage, are morphologically identical and similar to other phage described to date [39-41]. They all have icosahedral heads (60-65 nm x 60-70 nm), short tails (14-20 nm; Figure 1), and belong to the Podoviridae family. Only minor differences are visible in the composition of structural proteins of Brucella phage