Actinomyces pyogenes isolates from Sheep : Biochemical identification and confirmation by molecular method

Arcanobacterium (Actinomyces) pyogenes, are animal pathogens, they produce hemolytic exotoxin, pyolysin (PLO) and are opportunistic pathogens of economically important livestock such as dairy, beef cattle and sheep. Samples were collected from pleural fluid of 10 diseased and 10 healthy sheep. Samples were from neighboring village to gazelle breeding center, Riyadh Saud Arabia. The objectives of this study were to investigate A. pyogenes in these sheep. The isolates were grown on brain heart infusion (BHI; Difco) agar plates, supplemented with 5% bovine blood at 37°C and 5% CO2. Biochemical identification of 20 isolates revealed beta-hemolytic on blood agar, hydrolyzed gelatin, starch, were unable to hydrolyze esculin or reduce nitrate and did not produce urease. DNA was extracted from isolates using DNeasy Blood and Tissue Kits (Qiagen, NY, USA). Molecular tool using 16S rdeoxyribonucleic acid (DNA) universal eubacterial forward primer 16F27 (5 ́-AGA GTT TGA TCC TGG CTC AG-3 ́) and reverse primer 16SrDNA R1525 (5 ́-AAG GAG GTG ATC CAG CCG CA-3 ́), MWG Biotech AG (Ebersberg, Germany) identified the isolates successfully as A. pyogenes.


INTRODUCTION
Archanobacterium pyogenes is an anaerobic Grampositive bacilli and it is a part of the normal flora in many domestic animals (Carlos et al., 2009).In investigation of bacteria in human samples, polymerase chain reaction (PCR) based on bacterial 16S r-ribonucleic acid (RNA) genes were used by Fahriye et al. (2011).However, regular screening of bacterial population with Gram stain method, using Nugent's criteria which are reliable, easy to perform and well suited for the routine clinical laboratory has also been recommended by Manigeh et al. (2011).A previous study had attempted to analyze the Abbreviations: PLO, Pyolysin; DNA, deoxyribonucleic acid; PCR, polymerase chain reaction; RNA, ribonucleic acid; HUS, hemolytic uremic syndrome; BHI, brain heart infusion; SEM, scanning electron microscope; TSB, trypticase soy broth medium; TEM, transmission electron microscope; ELISA, enzyme-linked immunosorbent assay.effects of different levels of Aloe vera gel as an alternative to antibiotic, on performance and ileum morphology in broilers (Babak et al., 2011).The usefulness of antibiogram investigation of organism in clinical samples by disk-agar method which is very important for determining the susceptibility and resistance of microorganism in clinical samples was applied and reported by Lida et al. (2011).
The use of deoxyribonucleic acid (DNA)-based information (marker assisted selection and gene assisted selection, MAS and GAS, respectively) in conjunction with traditional selection methods could be useful to accelerate genetic progress for litter size and female reproduction efficiency in pigs (Stefania et al., 2011).Established multiplex-PCR assays study, for differentiation of methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant coagulase-negative Staphylococci, methicillin-sensitive coagulase-negative staphylococci and non-staphylococci strains, had been developed and applied by Zhenbo et al. (2011).Genetic distance within and between two natural population of Haloxylon saicornicum in Saudi Arabia was investigated at the DNA level by analysis of RAPD fragments (Fahd and Al-Qurainy, 2007).Further more, the use of molecular marker for identification of protected species offered a great promise in the field of conservation of biology (Ibrahim et al., 2011).Previous study by Liu et al. (2009) detected and identified gene cassettes of Arcanobacterium (Actinomyces) pyogenes isolates from cows with endometritis by using PCR.Accurate identification of bacterial isolates is an essential task in clinical microbiology.The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic method of 16S rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques.16S rDNA sequencing is a recent method of identification which offers a useful alternative (Mignard and Flandrois, 2006).Multiplex PCR technique was used to identify strains of shiga toxin-producing E. coli (STEC) and S. dysenteriae Type1 that have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans (Eisa et al., 2011).The objectives of this study was to investigate A. pyogenes in infected sheep of neighboring village to gazelle breeding center, Riyadh Saud Arabia.In this study primers specific for 16S rDNA in PCR reaction using template DNA from isolates were applied.

METHODS
Specimens were collected from pleural fluid of 10 infected and 10 healthy sheep.Kept in cold and transferred directly to the lab within 1 h for analysis.Enrichment and isolation of A. pyogenes was carried out following the method reported by Ertas et al. (2003).Pleural fluid samples were serially diluted in sterile saline solution up to 10 -4 and then collected Swabs from infected and healthy Shair 1119 sheep were inoculated in triplicates on 5% sheep blood agar medium and BHI agar and incubated aerobically in 5% CO2 at 37˚C for 72-96 h.The obtained single colonies were sub-cultured several times in fresh agar plates until pure homogeneous single colonies were obtained.The cells of the isolated bacteria were grown in the Trypticase Soy Broth medium (TSB) supplemented with 5% fetal bovine serum and incubated at 37°C for 18 h.Identification of the isolated bacteria was carried out using conventional biochemical methods.Cell morphology and cell wall type were examined using conventional gram stain.The cell morphology of the bacterial isolates was examined under SEM available in the Electron Microscopy unit in the National Research Center, Faculty of Science, King Saud University.API Coryne kits were used following the procedures of strip API guideline (France) and results were interoperated according to the manufacturer's instructions of the API guideline and finally the results were confirmed by molecular methods using universal eubacterial forward primer 16F27 (5´-AGA GTT TGA TCC TGG CTC AG-3´) and reverse primer 16R1525 (5´-AAG GAG GTG ATC CAG CCG CA-3´), MWG Biotech AG (Ebersberg, Germany).Total bacterial DNA was extracted using DNeasy Blood and Tissue Kits (Qiagen, NY, USA.Extracted DNA was electrophoresised with 1 ×TAE in agarose gel, by loading (12 µl) mixed with 3 µl of loading dye solution (Qiagen, NY, USA) into the gel wells.1kb DNA ladder (Roche, Mannheim, Germany) was used as the molecular size standard.The electrophoresis was run at 120 V for about 45 min.The gel was removed carefully from the gel tray and placed in ethidium bromide solution (1 µg/ml) for 30 min.And then was placed on an ultra violet transilluminator to visualize the DNA.The 16S-rDNA gene was amplified by PCR using universal eubacterial forward primer 16F27 (5´-AGA GTT TGA TCC TGG CTC AG-3´) and reverse primer 16R1525 (5´-AAG GAG GTG ATC CAG CCG CA-3´) derived from E. coli 16S-rDNA sequence synthesized by MWG Biotech AG (Ebersberg, Germany).
The PCR amplification was performed using the purified genomic DNA as a template.The PCR reaction contained, Forward primer, 1 μ (10 pmol/μl), reverse primer, 1 μ (10 pmol/μl), deoxynucleoside triphosphates 0.6 μl (10 mmol/liter) , l X PCR buffer 3 μl, MgCl 1.8 μl (25 mM), Taq polymerase 0.2 μl (5 U/μl), DNA template 2.5 μl (100 ng), Sterile distilled water 19.9 μl.The PCR reaction was performed in a peltier thermal cycler (BioRad, USA) using the following conditions: Initial denaturation (95°C, 5 min), denaturation (95°C, 30 s), annealing (52°C, 30 s), extension (70°C, 1.5 min), final extension (70°C, 5 min), and end/store forever (4°C).Agarose gel electrophoresis of the PCR products was carried by mixing 12 µl of it with 3 µl of loading dye solution (Qiagen.NY, USA) and were separated in a 1 % (w/v) agarose gel electrophoresis at 120 V in 1μ ×TAE buffer.A 1kb DNA ladder (Roche, Mannheim, Germany) was used as the molecular size standard.The gel was placed in ethidium bromide solution (1 µg/ml) for 30 min and was placed on an ultra violet transilluminator to visualize the DNA.The PCR product extraction from the agarose gel was curried by washing the gel twice with sterile distilled water and placed on an ultra violet transilluminator to visualize the DNA and then the amplified 16S-rDNA products were sliced off from the agarose gel with sterile razor blade.16SrDNA was obtained from the sliced gels, by using QIAquick gel Extraction Kits.

RESULTS AND DISCUSSION
Postmortem examination of the infected gazelle's lungs revealed similar supportive lesions in all of the infected sheep.Enrichment and isolation of A. pyogenes from the collected samples resulted in isolation of 20 bacterial isolates.The isolates showed colony surrounded with narrow sharp hemolytic zone.Furthermore, cell   morphology and size were examined under SEM and transmission electron microscope (TEM) (Figure 1 and 2).API strip identified all isolates as A. pyogenes.The results indicated that all isolates were non-motile, betahemolytic on blood agar, able to hydrolyze gelatin and starch, in addition, the isolates were unable to hydrolyze esculin or reduce nitrate and did not produce urease.16S rDNA was amplified by PCR successfully (Figure 3).The amplified 16S rDNA was sliced off from the gel and visualized under UV light (Figure 4).Biochemical characteristics of A. pyogenes isolated from healthy and infected sheep is presented in Table 1.Antibiogrammes were carried out for the 11 A. pyogenes isolates against 14 antibiotics presented in Table 2. A. pyogene DNA was isolated and Purified as shown in Figure 5. Antibiogrammes of 11 A. pyogenes isolates against 14 antibiotics were carried (Figure 6).urogenital tracts of a number of domestic animal species (Carlos et al., 2009).
The objective of this study was to investigate A. Pyogenes in suspected sheep kept in neighboring area to gazelle breeding center in Riyadh Saudi Arabia.Investigation of the relationship between symptoms and infection by A. Pyogenes, cell morphology and size were examined under SEM and TEM.Cells were about 0.8-1.0 and 0.3 μm in length and diameter respectively.The isolated bacterial strains were subjected to identification by biochemical testes and API Coryne strip.The biochemical tests revealed that all isolates were betahemolytic on blood agar, able to hydrolyze gelatin and starch, in addition, the isolates were unable to hydrolyze esculin or reduce nitrate and did not produce urease.
However there was some variation in sugar fermentation ability of different isolates.Collected pleural fluid samples from infected sheep indicated that A. pyogenes was detected in 85% of the infected sheep (17 out of 20).
Recently, a number of assays such as cell culture method, enzyme-linked immunosorbent assay (ELISA), reversed passive latex agglutination (RPLA), hybridization and PCR methods are available for the detection of bacterial infection.The cell culture method is sensitive but this approach is laborious, time-consuming and requires several days to identify the pathogen.The ELISA method is sensitive and rapid diagnostic tool but it requires monoclonal antibodies which are expensive.Hybridization procedure is a very sensitive and effective molecular technique for specific detection of the biological agents, according to their genetical structure.However, it is effectively used for the identification of pathogen, but it has got some limitations, including the risk of the using radioactive probes, complexity, time-consuming and the lack of its efficiency for some clinical samples in medical diagnostic laboratories.PCR assay is an effective method for the detection of wide range of genes and biological agents.This technique does not have the described limitations in the other methods.Therefore, these described time consuming or expensive procedures for identification of pathogen can be replaced with a PCR reaction (Eisa et al., 2011).In our study of A. pyogenes, PCR using DNA template of the isolates and specific primers for 16S rDNA was used for further confirmation of the pathogen.PCR products were analyzed using agarose gel electrophoresi and purified from the agarose gel. A. pyogenes is currently widespread pathogens throughout the world and has prompted a heightened interest and concern for the rapid detection of these pathogens as well as its related anti-biotic resistance determinant.Thus, rapid and accurate detection approaches are needed to reduce risk infection caused by A. pyogenes.This study was aimed to establish simple and rapid testing methods based on biochemical test and 16S rDNA PCR assays for identification of A. pyogene.No false positive amplification was observed, indicating the high specificity of the established PCR assay.

Conclusion
Number of assays, such as cell culture method, ELISA, RPLA, hybridization and PCR methods are available for the detection of bacterial infection as well as its toxins.The cell culture method is sensitive but this approach is laborious, time-consuming and requires several days to identify the pathogen.The ELISA method is sensitive and rapid diagnostic tool but it requires monoclonal antibodies which are expensive.Hybridization procedure is a very sensitive and effective molecular technique for specific detection of the biological agents, according to their genetically structure.However, it is effectively used for the identification of pathogen, but it has got some limitations, including the risk of the using radioactive probes, complexity, time-consuming and the lack of its efficiency for some clinical samples in medical diagnostic laboratories.PCR assay is an effective method for the detection of wide range of genes and biological agents.This technique does not have the described limitations in the other methods.Therefore, we attempted to combine classical biochemical method assays with PCR to avoid these described time consuming or expensive procedures for identification of pathogen.In our study of A. pyogenes, no false positive amplification was observed, indicating the high specificity of the established PCR assay.In conclusion, this established 16S rDNA PCR assay was demonstrated to be useful and powerful tools for the rapid detection of A. pyogene and the designed primers were specific for the identification of A. pyogenes and hence can be helpful to diagnosis of clinical samples.Further investigation should focus on the application to a large scale of clinical samples and comparative sensitivity and specificity with PCR-based.

Figure 1 .
Figure 1.Scanning electron microscope of the isolated Actinomyces pyogenes.

Figure 2 .
Figure 2. Transmission electron microscope of the isolated Actinomyces pyogenes.

Figure 4 .
Figure 4. Shows purified bacterial DNA.12 µl of the extracted bacterial DNA and 3 µl of the loading dye were mixed and separated in 0.8% agarose in 1x TAE buffer.The gel was placed ethidium bromide solution (1 µg/ml) for 30 min and visualized using UV tans illuminator.M: 1 kb DNA ladder.Lanes 1 to 11 isolates.

Figure 5 .
Figure 5.The amplified 16S-rDNA products being sliced off from the agarose gel for purification using QIAquick gel extraction kit.

Figure 6 .
Figure 6.Percentage of antibiotics resistant A. pyogenes isolates.Antibiogrammes were carried out for the 11 A. pyogenes isolates against 14 various antibiotics belonging to 9 antibiotic classes.

Table 1 .
Biochemical characteristics of A. pyogenes isolated from healthy and infected sheep.

Table 2 .
Summary of antibiotics susceptibility of isolated A. pyogenes strains.