Synergistic Effect of Bacteriophage and Antibiotic against Antibiotic-Resistant Salmonella Typhimurium

Kantiya Petsong,Kitiya Vongkamjan1, Juhee Ahn 한국식품위생안전성학회 2020 한국식품위생안전성학회지 Vol.35 No.2

본 연구는 항생제 내성 Salmonella Typhimurium CCARM 8009을 저해하기 위한 phage와 항생제 조합처리의 효과를 평가하였다. 디스크 확산법과 액체배지 희석법에 의해 phage와 항생제의 상승 저해효과를 측정하였고 배양을 통한 항생제 내성 유도를 평가하였다. Phage를 처리한 cefotaxime, chloramphenicol, ciprofloxacin, erythromycin의 디스크의 저해 구역은 각각 13.6%, 19.3%, 12.7%, 78.8% 로 증가되었다. Phage와 항생제 조합 처리에 의해 tetracycline, chloramphenicol, ciprofloxacin, erythromycin, streptomycin의 최소생육억제농도는 각각 64, 4, 0.0078, 64, 256 mg/mL으로 감소되었다. Phage와 항생제의 조합 처리는 항생제 내성 S. Typhimurium CCARM 8009을 효과적으로 저해하였다 (4 log reduction). 본 결과는 phage와 항생제의 조합처리는 항생제 내성균을 제어하기 위한 방법으로 충분히 응용가치가 높음을 보여주고 있다. In this study, we investigated the efficacy of Salmonella phage P22 combined with antibiotics to inhibit antibiotic-resistant S. Typhimurium CCARM 8009. The synergistic effect of phage P22 and antibiotics was evaluated by using disk diffusion and broth dilution assays. The development of Antimicrobial resistance was determined after time-kill assay. The antibiotic susceptibility assay showed the inhibition zone sizes around the antibiotic disks were increased up to 78.8% in the presence of phage (cefotaxime; 13.6%, chloramphenicol; 19.3%, ciprofloxacin; 12.7% and erythromycin; 78.8%). The minimum inhibitory concentration values of the combination treatment significantly decreased from 256 to 64 mg/mL for tetracycline, 8 to 4 mg/mL for chloramphenicol, 0.0156 to 0.0078 mg/mL for ciprofloxacin, 128 to 64 mg/mL for erythromycin and 512 to 256 mg/mL for streptomycin. The number of S. Typhimurium CCARM 8009 was approximately 4-log lower than that of the control throughout the combination treatment with phage P22 and ciprofloxacin delete at 37oC for 20 h. The results indicate that the development of antimicrobial resistance in S. Typhimurium could be reduced in the presence of phage treatment. This study provides promising evidence for the phage-antibiotic combination as an effective treatment to control antibiotic-resistant bacteria.

Bacteriophages of Pseudomonas tolaasii for the Biological Control of Brown Blotch Disease

Min-Hee Kim,Seong-Wan Park,Young-Kee Kim 한국응용생명화학회 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.1

Pseudomonas tolaasii causes brown blotch disease in cultivated mushrooms by producing tolaasin, a peptide toxin, which forms pores on the membrane and disrupts the cellular and fruiting body structures of mushrooms. For the biological control of this mushroom disease, virulent bacteriophages of P. tolaasii were isolated from the sewage of Cheongju, Korea. Twenty-one phages were isolated from four different locations, and their toxicities to host bacteria were measured by inspecting the turbidity and size of their plaques. They were divided into three categories on the basis of their toxicities to host bacteria. In order to test if these phages can be used for the biological control of mushroom diseases, a pitting test was performed. The surfaces of mushroom caps were inoculated with both pathogenic bacteria and their phages. Phage toxicity was analyzed by measuring the size of the blotches that formed on the surface of mushrooms, because these sizes are representative of the amount of tolaasin peptide produced by pathogenic bacteria in the presence of bacteriophages. The formation of blotches was completely blocked by co-incubated phages. These results show that phages can sterilize pathogenic bacteria in mushroom tissues as well as be useful for the biological control of brown blotch disease. The optimum conditions for the bactericidal activity of the phages were also determined. Pseudomonas tolaasii causes brown blotch disease in cultivated mushrooms by producing tolaasin, a peptide toxin, which forms pores on the membrane and disrupts the cellular and fruiting body structures of mushrooms. For the biological control of this mushroom disease, virulent bacteriophages of P. tolaasii were isolated from the sewage of Cheongju, Korea. Twenty-one phages were isolated from four different locations, and their toxicities to host bacteria were measured by inspecting the turbidity and size of their plaques. They were divided into three categories on the basis of their toxicities to host bacteria. In order to test if these phages can be used for the biological control of mushroom diseases, a pitting test was performed. The surfaces of mushroom caps were inoculated with both pathogenic bacteria and their phages. Phage toxicity was analyzed by measuring the size of the blotches that formed on the surface of mushrooms, because these sizes are representative of the amount of tolaasin peptide produced by pathogenic bacteria in the presence of bacteriophages. The formation of blotches was completely blocked by co-incubated phages. These results show that phages can sterilize pathogenic bacteria in mushroom tissues as well as be useful for the biological control of brown blotch disease. The optimum conditions for the bactericidal activity of the phages were also determined.

Pseudomonas tolaasii 박테리오파지에 특이적인 다클론항체 형성 및 이를 이용한 파지 교차 반응성

윤영배,박수진,김영기 한국응용생명화학회 2019 Journal of Applied Biological Chemistry (J. Appl. Vol.62 No.3

Pseudomonas tolaasii는 느타리버섯에 갈반병을 일으키는 병원균주로, 다양한 변이균주들을 분리하여 P1α와 P1β, P1γ 세 가지 소그룹으로 분류하였다. 각 그룹별 균주들에 특이적인 박테리오파지를 이용한 파지테라피는 갈반병 방제에 매우 성공적이었다. 본 연구에서는, 박테리오파지들의 특성을 구명하기 위하여 소그룹별 대표균주을 이용하여 파지를 분리하였고, 이들의다클론항체를 제작하여 파지들 사이에 유연관계를 조사하였다. 파지 준비물은 1010 pfu/mL 이상으로 토끼의 다리 근육에 주사하였고, 3회의 반복주사에 의해 다클론항체가 얻어졌다. 파지φ6264에 대한 항체의 역가는 2×107 Ab/mL 이상, 파지 φHK2 에 대해서는 1×106 Ab/mL, 파지 φHK19와 φHK23에 대해서는 1×107 Ab/mL 이상이었다. 항체와 이에 특이적인 파지 사이에는 매우 높은 반응특이성이 있었고, 소그룹이 다른 파지의 항체와 파지 사이에도 일부 교차반응성을 확인하였다. 파지 φ6264 에서 생성된 Abφ6264는 모든 P1α 소그룹의 파지들과 반응성을 보였으나, 파지 φHK16을 제외한 다른 소그룹의 파지들과는반응하지 않았다. P1γ 소그룹에서 생성된 AbφHK23은 P1β 소그룹의 모든 파지들을 불활성화시켜 가장 넓은 항체범위를 보였다. 항체와 파지를 이용한 숙주균과의 관계를 분석하였을 때, 16S rRNA 유전자 분석에 의한 숙주균의 근연관계와 항체를 이용한 숙주균의 파지들 사이의 구조적 근연관계는 상당히 차이가있음을 확인하였다. 결론적으로, 박테리오파지의 숙주균 특이성과 항체를 이용해 측정한 파지의 껍질단백질 구조유사성 사이에는 약한 상관성을 보였다. Pseudomonas tolaasii causes brown blotch disease on the oyster mushroom (Pleurotus ostreatus). Various pathogenic strains of P. tolaasii were isolated and divided into three subtypes, P1α, P1β, and P1γ. For phage therapy, bacteriophages against to these subtype strains were applied to mushroom cultivation and very successful to prevent from the disease. In this study, bacteriophages were isolated against the representative strains of subtype pathogens and their polyclonal antibodies were synthesized to investigate structural relationship among capsid proteins of phages. Phage preparations over 1010 pfu/mL were injected to rabbit thigh muscle and polyclonal antibodies were obtained after three times of boost injection. Titers of the antibodies obtained were over 2×107 Ab/mL for the phage φ6264, 1×106 Ab/mL for the phage φHK2, and 1×107 Ab/mL for the phage φHK19 and phage φHK23. High specific activities were observed between antibodies and the corresponding bacteriophages. Some crossreactivities between the antibodies and non-corresponding bacteriophages were also measured. Antibody Abφ6264 inactivated all phages of P1α subtype and only phage φHK16 among P1β subtype phages. Antibody AbφHK23 of P1γ subtype neutralized all phages of P1β subtype as well as the phage φHK23, showing the widest phage-inactivation range. When the structural-similarity studies of phages were investigated by using phage antibodies, closeness obtained by phylogenetic analysis of 16S rRNA genes of pathogenic strains were quite different from that of polyclonal antibody-specific structural similarity of phage capsid proteins. In conclusion, there is weak correlation between the host strain specificity of bacteriophage and its capsid structural similarity measured by phage antibodies.

Bacteriophages of Pseudomonas tolaasii for the Biological Control of Brown Blotch Disease

Kim, Min-Hee,Park, Seong-Wan,Kim, Young-Kee The Korean Society for Applied Biological Chemistr 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.1

Pseudomonas tolaasii causes brown blotch disease in cultivated mushrooms by producing tolaasin, a peptide toxin, which forms pores on the membrane and disrupts the cellular and fruiting body structures of mushrooms. For the biological control of this mushroom disease, virulent bacteriophages of P. tolaasii were isolated from the sewage of Cheongju, Korea. Twenty-one phages were isolated from four different locations, and their toxicities to host bacteria were measured by inspecting the turbidity and size of their plaques. They were divided into three categories on the basis of their toxicities to host bacteria. In order to test if these phages can be used for the biological control of mushroom diseases, a pitting test was performed. The surfaces of mushroom caps were inoculated with both pathogenic bacteria and their phages. Phage toxicity was analyzed by measuring the size of the blotches that formed on the surface of mushrooms, because these sizes are representative of the amount of tolaasin peptide produced by pathogenic bacteria in the presence of bacteriophages. The formation of blotches was completely blocked by co-incubated phages. These results show that phages can sterilize pathogenic bacteria in mushroom tissues as well as be useful for the biological control of brown blotch disease. The optimum conditions for the bactericidal activity of the phages were also determined.

Assessment of antibiotic resistance in bacteriophage-insensitive <i>Klebsiella pneumoniae</i>

Uddin, Md Jalal,Kim, Beomseok,Dawan, Jirapat,Ding, Tian,Kim, Jin-Chul,Ahn, Juhee Academic Press 2019 Microbial Pathogenesis Vol. No.

<P><B>Abstract</B></P> <P>This study was design to evaluate the physiological properties of bacteriophage-insensitive <I>Klebsiella pneumoniae</I> (BIKP) mutants in association with the antibiotic cross-resistance, β-lactamase activity, and gene expression. <I>Klebsiella pneumoniae</I> ATCC 23357(KP<SUP>WT</SUP>), ciprofloxacin-induced antibiotic-resistant <I>K</I>. <I>pneumoniae</I> ATCC 23357 (KP<SUP>CIP</SUP>), and clinically isolated antibiotic-resistant <I>K</I>. <I>pneumoniae</I> 10263 (KP<SUP>CLI</SUP>) were used to isolate BIKP mutants against KPB1, PBKP02, PBKP21, PBKP29, PBKP33, and PBKP35. PBKP35-induced mutants, including bacteriophage-insensitive <I>K</I>. <I>pneumoniae</I> ATCC 23357 (BIKP<SUP>WT</SUP>), ciprofloxacin-induced <I>K. pneumoniae</I> ATCC 23357 (BIKP<SUP>CIP</SUP>), and clinically isolated antibiotic-resistant <I>K. pneumoniae</I> CCARM 10263 (BIKP<SUP>CLI</SUP>). BIKP<SUP>WT</SUP>, BIKP<SUP>CIP</SUP>, and BIKP<SUP>CLI</SUP> were resistant to <I>Klebsiella</I> bacteriophages, KPB1, PBKP02, PBKP21, PBKP29, and PBKP33. The antibiotic cross-resistance to cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, levofloxacin, and nalidixic acid was observed in BIKP<SUP>WT</SUP>. The relative expression levels of <I>vagC</I> was increased by more than 8-folds in BIKP<SUP>WT</SUP>, corresponding to the increased β-lactamase activity. The <I>aac(6′)-Ib-cr</I> was overexpressed in BIKP mutants, responsible for aminoglycoside and quinolone resistance. The phage-resistant mutants decreased the antibiotic susceptibilities in association with β-lactamase activity and antibiotic resistance-related gene expression. The results pointed out the cross-resistance of BIKP mutants to antibiotics, which might be considered when applying for the therapeutic use of bacteriophage.</P>

Prediction and identification of new type holin protein of Escherichia coli phage ECP26

박도원,이종훈,박종현 한국식품과학회 2022 Food Science and Biotechnology Vol.31 No.7

Bacteriophages (phages) infecting specifically target bacteria utilize a unique lysis module known as the holin-endolysin cassette to release progeny. Studies on the phage lytic proteins could contribute to the development of alternatives to antibiotics. Here, we predicted and identified the holin protein of rV5-like phage ECP26 for increasing lytic activity of the phage endolysin. In silico analysis revealed that open reading frame 151 (ORF151) of ECP26 contained two transmembrane domains. Co-expression of endolysin with ORF151 resulted in the cell lysis of Escherichia coli, suggesting that ORF151 protein functioned as the holin that disrupted the cytoplasmic membrane. The putative holin showed a high amino acid homology by more than 80% to the predicted holins of rV5-like phages. Therefore, the holin protein would be helpful for developing efficient lysis strategies with endolysin against gram-negative E. coli.

Characterization of Phage-Resistant Strains Derived from Pseudomonas tolaasii 6264, which Causes Brown Blotch Disease

( Yeong-bae Yun ),( Ji-hye Han ),( Young-kee Kim ) 한국미생물 · 생명공학회 2018 Journal of microbiology and biotechnology Vol.28 No.12

Pseudomonas tolaasii 6264 is a representative strain that causes bacterial blotch disease on the cultivated oyster mushroom, Pleurotus ostreatus. Bacteriophages are able to sterilize the pathogenic P. tolaasii strains, and therefore, they can be applied in creating disease-free mushroom cultivation farms, through a method known as “phage therapy”. For successful phage therapy, the characterization of phage-resistant strains is necessary, since they are frequently induced from the original pathogenic bacteria in the presence of phages. When 10 different phages were incubated with P. tolaasii 6264, their corresponding phage-resistant strains were obtained. In this study, changes in pathogenic, genetic, and biochemical characteristics as well as the acquired phage resistance of these strains were investigated. In the phylogenetic analyses, all phage-resistant strains were identical to the original parent strain based on the sequence comparison of 16S rRNA genes. When various phage-resistant strains were examined by three different methods, pitting test, white line test, and hemolytic activity, they were divided into three groups: strains showing all positive results in three tests, two positive in the first two tests, and all negative. Nevertheless, all phage-resistant strains showed that their pathogenic activities were reduced or completely lost.

Synergistic Effect of Bacteriophage and Antibiotic against Antibiotic-Resistant Salmonella Typhimurium

Petsong, Kantiya,Vongkamjan, Kitiya,Ahn, Juhee The Korean Society of Food Hygiene and Safety 2020 한국식품위생안전성학회지 Vol.35 No.2

본 연구는 항생제 내성 Salmonella Typhimurium CCARM 8009을 저해하기 위한 phage와 항생제 조합처리의 효과를 평가하였다. 디스크 확산법과 액체배지 희석법에 의해 phage와 항생제의 상승 저해효과를 측정하였고 배양을 통한 항생제 내성 유도를 평가하였다. Phage를 처리한 cefotaxime, chloramphenicol, ciprofloxacin, erythromycin의 디스크의 저해 구역은 각각 13.6%, 19.3%, 12.7%, 78.8%로 증가되었다. Phage와 항생제 조합 처리에 의해 tetracycline, chloramphenicol, ciprofloxacin, erythromycin, streptomycin의 최소생육억제농도는 각각 64, 4, 0.0078, 64, 256 mg/mL으로 감소되었다. Phage와 항생제의 조합 처리는 항생제 내성 S. Typhimurium CCARM 8009을 효과적으로 저해하였다 (4 log reduction). 본 결과는 phage와 항생제의 조합처리는 항생제 내성균을 제어하기 위한 방법으로 충분히 응용가치가 높음을 보여주고 있다. In this study, we investigated the efficacy of Salmonella phage P22 combined with antibiotics to inhibit antibiotic-resistant S. Typhimurium CCARM 8009. The synergistic effect of phage P22 and antibiotics was evaluated by using disk diffusion and broth dilution assays. The development of Antimicrobial resistance was determined after time-kill assay. The antibiotic susceptibility assay showed the inhibition zone sizes around the antibiotic disks were increased up to 78.8% in the presence of phage (cefotaxime; 13.6%, chloramphenicol; 19.3%, ciprofloxacin; 12.7% and erythromycin; 78.8%). The minimum inhibitory concentration values of the combination treatment significantly decreased from 256 to 64 mg/mL for tetracycline, 8 to 4 mg/mL for chloramphenicol, 0.0156 to 0.0078 mg/mL for ciprofloxacin, 128 to 64 mg/mL for erythromycin and 512 to 256 mg/mL for streptomycin. The number of S. Typhimurium CCARM 8009 was approximately 4-log lower than that of the control throughout the combination treatment with phage P22 and ciprofloxacin delete at 37℃ for 20 h. The results indicate that the development of antimicrobial resistance in S. Typhimurium could be reduced in the presence of phage treatment. This study provides promising evidence for the phage-antibiotic combination as an effective treatment to control antibiotic-resistant bacteria.

Complete genome sequence of a newly isolated lytic bacteriophage, EFAP-1 of <i>Enterococcus faecalis</i>, and antibacterial activity of its endolysin EFAL-1

Son, J.S.,Jun, S.Y.,Kim, E.B.,Park, J.E.,Paik, H.R.,Yoon, S.J.,Kang, S.H.,Choi, Y.-J. Blackwell Publishing Ltd 2010 Journal of applied microbiology Vol.108 No.5

<P>Abstract</P><P>Aims: </P><P>In this work, we aimed to identify an effective treatment of infections caused by <I>Enterococcus</I> spp. strains resistant to conventional antibiotics.</P><P>Methods and Results: </P><P>We report the isolation and characterization of a new lytic bacteriophage, designated bacteriophage EFAP-1, that is capable of lysing <I>Enterococcus faecalis</I> bacteria that exhibit resistance to multiple antibiotics. EFAP-1 has low sequence similarity to all known bacteriophages. Transmission electron microscopy confirmed that EFAP-1 belongs to the <I>Siphoviridae</I> family. A putative lytic protein of EFAP-1, endolysin EFAL-1, is encoded in ORF 2 and was expressed in <I>Escherichia coli</I>. Recombinant EFAL-1 had broad-spectrum lytic activity against several Gram-positive pathogens, including <I>Ent. faecalis</I> and <I>Enterococcus faecium.</I></P><P>Conclusions: </P><P>The complete genome sequence of the newly isolated enterococcal lytic phage was analysed, and it was demonstrated that its recombinant endolysin had broad lytic activity against various Gram-positive pathogens.</P><P>Significance and Impact of the Study: </P><P>Bacteriophage EFAP-1 and its lytic protein, EFAL-1, can be utilized as potent antimicrobial agents against <I>Enterococcus</I> spp. strains resistant to conventional antibiotics in hospital infections and also as environmental disinfectants to control disease-causing <I>Enterococcus</I> spp. in dairy farms.</P>

Synergistic Antimicrobial Activity of Bacteriophages and Antibiotics against Staphylococcus aureus

조아라,Tian Ding,안주희 한국식품과학회 2016 Food Science and Biotechnology Vol.25 No.3

This study was designed to assess the synergistic antimicrobial effect of phages combined with antibiotics against Staphylococcus aureus. The phage-antibiotic synergy (PAS) effect was evaluated using the fractional inhibitory concentration (FIC) and flow cytometric analysis. The determined minimum inhibitory concentration (MIC) values varied from 0.125 to 128 μg/mL for S. aureus KACC 13236 (SAS) and from 0.25 to >256 μg/mL for S. aureus CCARM 3080 (SAR). The PAS effect was more pronounced in SAS treated with phage SA11 in the presence of cefoxitin (FIC=0.62), chloramphenicol (FIC=0.54), and polymyxin B (FIC=0.38). SAS and SAR cells were injured when exposed to asublethal concentration of ciprofloxacin, whereas these cells were highly susceptible to the phage-antibiotic combined treatment, showing 96% of relative percentages of injured and dead cells. The results suggest that the combined treatment of phages and antibiotics can be used to improve antimicrobial efficacy against antibiotic-resistant bacteria.