RESUMEN
The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.
Asunto(s)
Técnicas Biosensibles , Endopeptidasas , Staphylococcus aureus , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/virología , Técnicas Biosensibles/métodos , Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Endopeptidasas/genética , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Humanos , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/química , Fagos de Staphylococcus/aislamiento & purificación , Animales , Técnicas de Amplificación de Ácido Nucleico/métodos , Infecciones Estafilocócicas/microbiología , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Nucleasa Microcócica/química , Nucleasa Microcócica/metabolismo , Nucleasa Microcócica/genética , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
OBJECTIVE: This study aimed to introduce a lytic bacteriophage against Staphylococcus saprophyticus from wastewater in Gorgan, northern Iran. RESULTS: The vB_SsapS-46 phage was isolated from urban wastewater and formed round and clear plaques on bacterial culture. It was visualized by electron microscopy and had a large head (approximately 106 nm) and a long tail (approximately 150 nm), indicating that it belongs to the Siphoviridae family. The host range of vB_SsapS-46 was determined using a spot test on 35 S. saprophyticus clinical isolates, and it was able to lyse 12 of the 35 clinical isolates (34%). Finally, the relationship between phage sensitivity and adherence genes was assessed, revealing no significant correlation between phage sensitivity and the frequency of adherence genes. The vB_SsapS-46 phage can be used alone or in a mixture in future studies to control urinary tract infections caused by this bacterium, especially in the elimination of drug-resistant pathogens.
Asunto(s)
Fagos de Staphylococcus , Staphylococcus saprophyticus , Staphylococcus saprophyticus/virología , Staphylococcus saprophyticus/genética , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/aislamiento & purificación , Fagos de Staphylococcus/ultraestructura , Fagos de Staphylococcus/fisiología , Siphoviridae/genética , Siphoviridae/aislamiento & purificación , Siphoviridae/ultraestructura , Irán , Aguas Residuales/microbiología , Aguas Residuales/virología , Especificidad del Huésped , Humanos , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiologíaRESUMEN
Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%-1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications.
Asunto(s)
Genoma Viral , Fagos de Staphylococcus , Staphylococcus aureus , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/fisiología , Fagos de Staphylococcus/aislamiento & purificación , Staphylococcus aureus/virología , Staphylococcus aureus/genética , Humanos , Composición de Base , Podoviridae/genética , Podoviridae/aislamiento & purificación , Podoviridae/clasificación , Podoviridae/fisiología , Células HT29 , Tamaño del GenomaRESUMEN
Staphylococcus aureus can be a harmless coloniser, but it can also cause severe infections in humans, livestock and wildlife. Regarding the latter, only few studies have been performed and knowledge on virulence factors is insufficient. The aim of the present study was to study S. aureus isolates from deceased wild beavers (Castor fiber). Seventeen isolates from eleven beavers, found in Germany and Austria, were investigated. Antimicrobial and biocide susceptibility tests were performed. Isolates were characterised using S. aureus-specific DNA microarrays, spa typing and whole-genome sequencing. From two isolates, prophages were induced by mitomycin C and studied by transmission electron microscopy. Four isolates belonged to clonal complex (CC) 8, CC12, and CC398. Twelve isolates belonged to CC1956 and one isolate was CC49. The CC49 and CC1956 isolates carried distinct lukF/S genes related to the Panton-Valentine leukocidin (PVL) from human isolates of S. aureus. These genes were located on related, but not identical, Siphovirus prophages. The beavers, from which those isolates originated, suffered from abscesses, purulent organ lesions and necrotising pneumonia, i.e., clinical manifestations resembling symptoms of severe PVL-associated disease in humans. It might thus be assumed that the "Beaver Leukocidin (BVL, lukF/S-BV)"-positive strains are beaver-specific pathogens, and further studies on their clinical role as well as on a possible transmissibility to other species, including humans, are warranted.
Asunto(s)
Toxinas Bacterianas/análisis , Exotoxinas/análisis , Leucocidinas/análisis , Roedores/microbiología , Infecciones Estafilocócicas/microbiología , Fagos de Staphylococcus/aislamiento & purificación , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/virología , Animales , Toxinas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Exotoxinas/genética , Genes Bacterianos , Genes Virales , Humanos , Leucocidinas/genética , Infecciones Estafilocócicas/veterinaria , Fagos de Staphylococcus/genética , Staphylococcus aureus/genéticaRESUMEN
The host range of bacteriophages defines their impact on bacterial communities and genome diversity. Here, we characterize 94 novel staphylococcal phages from wastewater and establish their host range on a diversified panel of 117 staphylococci from 29 species. Using this high-resolution phage-bacteria interaction matrix, we unveil a multi-species host range as a dominant trait of the isolated staphylococcal phages. Phage genome sequencing shows this pattern to prevail irrespective of taxonomy. Network analysis between phage-infected bacteria reveals that hosts from multiple species, ecosystems, and drug-resistance phenotypes share numerous phages. Lastly, we show that phages throughout this network can package foreign genetic material enclosing an antibiotic resistance marker at various frequencies. Our findings indicate a weak host specialism of the tested phages, and therefore their potential to promote horizontal gene transfer in this environment.
Asunto(s)
Genoma Viral , Especificidad del Huésped , Fagos de Staphylococcus/genética , Staphylococcus/genética , Aguas Residuales , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Ecosistema , Transferencia de Gen Horizontal , Consorcios Microbianos/genética , Pruebas de Sensibilidad Microbiana , Filogenia , Staphylococcus/clasificación , Staphylococcus/efectos de los fármacos , Staphylococcus/virología , Fagos de Staphylococcus/clasificación , Fagos de Staphylococcus/aislamiento & purificación , Aguas Residuales/microbiología , Aguas Residuales/virología , Microbiología del AguaRESUMEN
The majority of previously described Staphylococcus aureus bacteriophages belong to three major groups, namely, P68-like podophages, Twort-like or K-like myophages, and a more diverse group of temperate siphophages. Here, we present the following three novel S. aureus "jumbo" phages: MarsHill, Madawaska, and Machias. These phages were isolated from swine production environments in the United States and represent a novel clade of S. aureus myophage. The average genome size for these phages is â¼269 kb with each genome encoding â¼263 predicted protein-coding genes. Phage genome organization and content are similar to those of known jumbo phages of Bacillus sp., including AR9 and vB_BpuM-BpSp. All three phages possess genes encoding complete virion and nonvirion RNA polymerases, multiple homing endonucleases, and a retron-like reverse transcriptase. Like AR9, all of these phages are presumed to have uracil-substituted DNA which interferes with DNA sequencing. These phages are also able to transduce host plasmids, which is significant as these phages were found circulating in swine production environments and can also infect human S. aureus isolates. IMPORTANCE This study describes the comparative genomics of the following three novel S. aureus jumbo phages: MarsHill, Madawaska, and Machias. These three S. aureus myophages represent an emerging class of S. aureus phage. These genomes contain abundant introns which show a pattern consistent with repeated acquisition rather than vertical inheritance, suggesting intron acquisition and loss are active processes in the evolution of these phages. These phages have presumably hypermodified DNA which inhibits sequencing by several different common platforms. Therefore, these phages also represent potential genomic diversity that has been missed due to the limitations of standard sequencing techniques. In particular, such hypermodified genomes may be missed by metagenomic studies due to their resistance to standard sequencing techniques. Phage MarsHill was found to be able to transduce host DNA at levels comparable to that found for other transducing S. aureus phages, making it a potential vector for horizontal gene transfer in the environment.
Asunto(s)
Genoma Viral , Myoviridae/genética , Fagos de Staphylococcus/genética , Staphylococcus aureus/virología , Animales , ADN Viral/genética , ARN Polimerasas Dirigidas por ADN/genética , Genómica , Intrones , Myoviridae/aislamiento & purificación , Myoviridae/fisiología , Myoviridae/ultraestructura , Análisis de Secuencia de ADN , Fagos de Staphylococcus/aislamiento & purificación , Fagos de Staphylococcus/fisiología , Fagos de Staphylococcus/ultraestructura , Porcinos , Transducción Genética , Proteínas Virales/genéticaRESUMEN
Staphylococcus aureus infections are of growing concern given the increased incidence of antibiotic resistant strains. Egypt, like several other countries, has seen alarming increases in methicillin-resistant S. aureus (MRSA) infections. This species can rapidly acquire genes associated with resistance, as well as virulence factors, through mobile genetic elements, including phages. Recently, we sequenced 56 S. aureus genomes from Alexandria Main University Hospital in Alexandria, Egypt, complementing 17 S. aureus genomes publicly available from other sites in Egypt. In the current study, we found that the majority (73.6%) of these strains contain intact prophages, including Biseptimaviruses, Phietaviruses, and Triaviruses. Further investigation of these prophages revealed evidence of horizontal exchange of the integrase for two of the prophages. These Egyptian S. aureus prophages are predicted to encode numerous virulence factors, including genes associated with immune evasion and toxins, including the Panton-Valentine leukocidin (PVL)-associated genes lukF-PV/lukS-PV. Thus, prophages are likely to be a major contributor to the virulence of S. aureus strains in circulation in Egypt.
Asunto(s)
Profagos/aislamiento & purificación , Fagos de Staphylococcus/aislamiento & purificación , Staphylococcus aureus/virología , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Egipto , Humanos , Profagos/clasificación , Profagos/genética , Infecciones Estafilocócicas/microbiología , Fagos de Staphylococcus/clasificación , Fagos de Staphylococcus/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , VirulenciaRESUMEN
BACKGROUND: Staphylococcus aureus is the causative agent of chronic mastitis, and can form a biofilm that is difficult to completely remove once formed. Disinfectants are effective against S. aureus, but their activity is easily affected by environmental factors and they are corrosive to equipment and chemically toxic to livestock and humans. Therefore, we investigated the potential utility of a bacteriophage as a narrow-spectrum disinfectant against biofilms formed by S. aureus. In this study, we isolated and characterized bacteriophage vB_SauM_SDQ (abbreviated to SDQ) to determine its efficacy in removing S. aureus biofilms. RESULTS: SDQ belongs to the family Myoviridae and consists of a hexagonal head, long neck, and short tail. This phage can sterilize a 109 CFU/mL culture of S. aureus in 12 h and multiply itself 1000-fold in that time. Biofilms formed on polystyrene, milk, and mammary-gland tissue were significantly reduced after SDQ treatment. Fluorescence microscopy and scanning electron microscopy showed that SDQ destroyed the biofilm structure. Moreover, the titer of SDQ remained relatively high after the lysis of the bacteria and the removal of the biofilm, exerting a continuous bacteriostatic effect. SDQ also retained its full activity under conditions that mimic common environments, i.e., in the presence of nonionic detergents, tap water, or organic materials. A nonionic detergent (Triton X-100) enhanced the removal of biofilm by SDQ. CONCLUSIONS: Our results suggest that SDQ, a specific lytic S. aureus phage, can be used to control biofilm infections. SDQ maintains its full activity in the presence of nonionic detergents, tap water, metal chelators, and organic materials, and can be used in combination with detergents. We propose this phage as a narrow-spectrum disinfectant against S. aureus, to augment or supplement the use of broad-spectrum disinfectants in the prevention and control of the mastitis and dairy industry contamination caused by S. aureus.
Asunto(s)
Biopelículas , Mastitis/veterinaria , Myoviridae/aislamiento & purificación , Infecciones Estafilocócicas/prevención & control , Fagos de Staphylococcus/aislamiento & purificación , Staphylococcus aureus/virología , Animales , Bovinos , Industria Lechera , Desinfectantes , Femenino , Mastitis/microbiología , Mastitis/prevención & control , Mastitis/terapia , Microscopía Electrónica de Rastreo , Myoviridae/genética , Myoviridae/fisiología , Terapia de Fagos , Aguas del Alcantarillado/microbiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/terapia , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/fisiología , Staphylococcus aureus/fisiologíaRESUMEN
Two Staphylococcus aureus bacteriophages, KSAP7 and KSAP11, were isolated from sewage and characterized. Based on morphology and DNA sequences, they were assigned to the genus Silviavirus, subfamily Twortvirinae, family Herelleviridae, whose members are hypothesized to be suitable for bacteriophage therapy. The KSAP7 and KSAP11 genomes were 137,950 and 138,307 bp in size, respectively. Although their DNA sequences were almost identical, evidence of site-specific DNA rearrangements was found in two regions. Changes in the number of PIEPEK amino acid sequence repeats encoded by orf10 and the insertion/deletion of a 541-bp sequence that includes a possible tail-related gene were identified.
Asunto(s)
Caudovirales/genética , ADN Viral/genética , Genoma Viral , Filogenia , Fagos de Staphylococcus/genética , Staphylococcus aureus/virología , Secuencia de Aminoácidos , Caudovirales/clasificación , Caudovirales/aislamiento & purificación , Reordenamiento Génico , Tamaño del Genoma , Mutación INDEL , Japón , Sistemas de Lectura Abierta , Terapia de Fagos , Alineación de Secuencia , Fagos de Staphylococcus/clasificación , Fagos de Staphylococcus/aislamiento & purificaciónRESUMEN
Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 °C.Key points⢠A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products. ⢠Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing. ⢠SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus. ⢠Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.
Asunto(s)
Microbiología de Alimentos/métodos , Podoviridae/metabolismo , Fagos de Staphylococcus/aislamiento & purificación , Fagos de Staphylococcus/metabolismo , Staphylococcus aureus/efectos de los fármacos , Animales , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Pollos/virología , Peces/virología , Genoma Viral , Especificidad del Huésped , Nisina/farmacología , Podoviridae/genética , Podoviridae/aislamiento & purificación , Carne de Cerdo/virología , Fagos de Staphylococcus/genética , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/virología , Virulencia/efectos de los fármacosRESUMEN
In this study, the genome of a new strain of lytic Staphylococcus aureus Herelleviridae, vBSM-A1, was characterized and annotated. The phage was isolated from sewage samples collected in Xinjiang Province, China. The genome of vBSM-A1 was found to comprise a linear double-stranded DNA of 140,654 bp length, with a G + C content of 30.33%. A total of 215 ORFs were detected in the phage DNA, 74 of which were functionally assigned. The 3D structure model of endolysin LysK (ORF 143) was created using Phyre2.
Asunto(s)
Genoma Viral/genética , Fagos de Staphylococcus/genética , Composición de Base , China , ADN Viral/química , ADN Viral/genética , Endopeptidasas/química , Endopeptidasas/genética , Modelos Moleculares , Sistemas de Lectura Abierta , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Fagos de Staphylococcus/aislamiento & purificaciónRESUMEN
Brazil has the second-largest dairy cattle herd in the world, and bovine mastitis still can cause significant losses for dairy farmers. Despite this fact, little information is available about milk microbial composition of Brazilian dairy cows, as well as the potential use of bacteriophages in the control of S. aureus. Here, we investigated milk bacterial composition of 28 Holstein Fresian cows (109 teats), selected in the dry-off period, using 16S rRNA analysis. Furthermore, a representative S. aureus strain (UFV2030RH1) was obtained at drying-off for isolation of a bacteriophage (vB_SauM-UFV_DC4, UFV_DC4) and bacterial genomic comparison purposes. Our outcomes revealed that Staphylococcus was the third most prevalent genus and positively correlated with subclinical mastitis events. As a major finding, genomic analyses showed the presence of adhesive matrix molecules that recognize microbial surface components (MSCRAMM) in UFV2030RH1 and might indicate great biofilm formation capability. A minimum inhibitory concentration (MIC) assay showed that resistance to ampicillin was the highest among the antibiotic tested in S. aureus 3059 and UFV2030RH1, displaying values four and sixteen times greater than MIC resistance breakpoint, respectively. Together, our results suggest that Staphylococcus is highly prevalent in dairy cows at drying-off and the use of the phage UFV_DC4 as a biocontrol agent must be investigated in future studies.
Asunto(s)
Mastitis Bovina/microbiología , Leche/microbiología , ARN Ribosómico 16S/genética , Fagos de Staphylococcus/fisiología , Staphylococcus aureus/clasificación , Resistencia a la Ampicilina , Animales , Antibacterianos/farmacología , Bovinos , ADN Bacteriano/genética , ADN Ribosómico/genética , Femenino , Genómica , Mastitis Bovina/prevención & control , Filogenia , Análisis de Secuencia de ADN , Fagos de Staphylococcus/aislamiento & purificación , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/virologíaRESUMEN
The emergence of life-threatening methicillin-resistant Staphylococcus aureus (MRSA) has led to increased interest in the use of bacteriophages as an alternative therapy to antibiotics. The success of phage therapy is greatly dependent on the selected phage possessing a wide host range. This study describes phage ɸMR003 isolated from sewage influent at a municipal wastewater treatment plant in Tokyo, Japan. ɸMR003 could infect 97% of 104 healthcare- and community-associated MRSA strains tested, compared with 73% for phage ɸSA012, which has a broad host range against bovine mastitis S. aureus. Genome analysis revealed that ɸMR003 belongs to the genus Silviavirus which has not been studied extensively. ɸMR003 recognizes and binds to wall teichoic acid (WTA) of S. aureus during infection. In silico comparisons of the genomes of ɸMR003 and ɸSA012 revealed that ORF117 and ORF119 of ɸMR003 are homologous to the putative receptor-binding proteins ORF103 and ORF105 of ɸSA012, with amino acid similarities of 75% and 72%, respectively. ORF104, which is an N-acetylglucosaminidase found in the ɸMR003 tail, may facilitate phage's infection onto the WTA-null S. aureus RN4220. The differences in tail and baseplate proteins may be key contributing factors to the different host specificities of ɸMR003 and ɸSA012. ɸMR003 showed strong adsorptivity, but not infectivity, against S. aureus SA003, which may be influenced by the bacterium's restriction modification system. This study expands our knowledge of the genomic diversity and host specificity of Silviavirus, which is a potential phage therapy candidate for MRSA infections.
Asunto(s)
Genoma Viral , Especificidad del Huésped , Staphylococcus aureus Resistente a Meticilina/virología , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/fisiología , Variación Genética , Humanos , Terapia de Fagos , Aguas del Alcantarillado/virología , Infecciones Estafilocócicas/terapia , Fagos de Staphylococcus/aislamiento & purificación , Ácidos Teicoicos/metabolismo , Tokio , Acoplamiento ViralRESUMEN
A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it.
Asunto(s)
Citocinas/genética , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Células Dendríticas/virología , Expresión Génica , Fagos de Staphylococcus/fisiología , Staphylococcus aureus/virología , Citocinas/metabolismo , Células Dendríticas/inmunología , Especificidad del Huésped , Humanos , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/virología , Terapia de Fagos , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/terapia , Fagos de Staphylococcus/aislamiento & purificación , Replicación ViralRESUMEN
The bacteriophage K1/420 is a member of genus Kayvirus that was extensively studied as an alternative treatment to combat bacterial infections caused by antibiotic-resistant Staphylococcus aureus strains. Despite the promise of phage therapy, the development of clinical applications of phages is facing regulatory and technical hurdles before it can receive acceptance in the Western World. Suitable simple and accurate diagnostic techniques to control the quality of the phage, which would satisfy the requirements of regulatory authorities are still being discussed. Here, we present the conditions for the simultaneous separation and detection of phage K1/420 and S. aureus by CZE and by CIEF were found, and the phage isoelectric point was determined to be 3.6. After removing the cell debris, the phage was successfully purified from the crude phage lysate and pre-concentrated by preparative isoelectric focusing. Its zone was localized by the positions of colored pI markers in the cellulose bed. The phage from the harvested zone had a decreased ability to infect its host. However, it was suitable for its separation, detection and identification by capillary electrophoretic methods, MALDI-TOF MS and electron microscopy.
Asunto(s)
Focalización Isoeléctrica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Fagos de Staphylococcus/aislamiento & purificación , Fagos de Staphylococcus/fisiología , Staphylococcus aureus/virología , Humanos , Fagos de Staphylococcus/clasificaciónRESUMEN
Staphylococcus aureus is a commensal and pathogenic bacterium that causes infections in humans and animals. It is a major cause of nosocomial infections worldwide. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the pathogen are necessary. In this respect, polyvalent staphylococcal myoviruses have been demonstrated to be excellent candidates for phage therapy. Here we present the characterization of the bacteriophage vB_SauM-fRuSau02 (fRuSau02) that was isolated from a commercial Staphylococcus bacteriophage cocktail produced by Microgen (Moscow, Russia). The genomic analysis revealed that fRuSau02 is very closely related to the phage MSA6, and possesses a large genome (148,464 bp), with typical modular organization and a low G+C (30.22%) content. It can therefore be classified as a new virus among the genus Twortlikevirus. The genome contains 236 predicted genes, 4 of which were interrupted by insertion sequences. Altogether, 78 different structural and virion-associated proteins were identified from purified phage particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The host range of fRuSau02 was tested with 135 strains, including 51 and 54 Staphylococcus aureus isolates from humans and pigs, respectively, and 30 coagulase-negative Staphylococcus strains of human origin. All clinical S. aureus strains were at least moderately sensitive to the phage, while only 39% of the pig strains were infected. Also, some strains of Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus and Staphylococcus pseudointer were sensitive. We conclude that fRuSau02, a phage therapy agent in Russia, can serve as an alternative to antibiotic therapy against S. aureus.
Asunto(s)
Genoma Viral , Myoviridae/genética , Fagos de Staphylococcus/genética , Staphylococcus aureus/virología , Animales , Infección Hospitalaria , Especificidad del Huésped , Humanos , Microscopía Electrónica , Myoviridae/aislamiento & purificación , Myoviridae/ultraestructura , Terapia de Fagos , Federación de Rusia , Infecciones Estafilocócicas/terapia , Fagos de Staphylococcus/aislamiento & purificación , Fagos de Staphylococcus/ultraestructura , Porcinos , ViriónRESUMEN
A Staphylococcus aureus (S. aureus)-specific lytic bacteriophage P-S. aureus-9, isolated from an environmental water sample, was assembled on magnetic beads for capturing S. aureus from samples through magnetic separation. Horseradish Peroxidase (HRP) labeled immunoglobulin (IgG) antibodies were used to detect the captured S. aureus by reacting with protein A on S. aureus followed by colorimetric signals, which were generated from the catalytic reaction between HRP and the substrate 3,3',5,5'-Tetramethylbenzidine (TMB). Under optimal conditions, the calibration curve was linear from 1.0×104 to 1.0×106CFUmL-1. The limit of detection (LOD) for the assay was 2.47×103CFUmL-1 and 8.86×103CFUmL-1 of S. aureus in PBS and apple juice, respectively. Moreover, the whole assay revealed outstanding specificity towards S. aureus, without any interference of common pathogenic bacteria, and can be completed within 90min without any pre-enrichment. As far as known, it was the first time to detect S. aureus based on the double site recognition of bacteriophage and mammal IgG. The novel approach has shown good potentials for a rapid, specific, cheap and simple detection of S. aureus in food samples.
Asunto(s)
Jugos de Frutas y Vegetales/microbiología , Infecciones Estafilocócicas/microbiología , Fagos de Staphylococcus/aislamiento & purificación , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/virología , Bencidinas/química , Técnicas Biosensibles/métodos , Humanos , Inmunoensayo/métodos , Inmunoglobulina G/química , Separación Inmunomagnética/métodos , Límite de Detección , Malus/microbiologíaRESUMEN
BACKGROUND: The implementation of phage therapy is re-emerging with the increase in widespread antibiotic-resistant bacteria. METHODS: Staphylococcus phage JD007 was characterized and its complete genome sequence analysed. RESULTS: Staphylococcus phage JD007 was classified as belonging to the Myoviridae family based on its morphology, as observed by transmission electron microscopy. Its lytic activity was stable between pH 5-11 and below 42 °C; moreover, an absorbance curve showed that nearly 90% of the viral particles had adsorbed to its host after a 20 min co-incubation. The complete genome size is 141,836 bp, making JD007 one of the largest Staphylococcus phages of Myoviridae. No identifiable resistance or virulence genes were found in the JD007 genome. JD007 was able to lyse 95% of S. aureus isolates, including the prevalent ST239-MRSA and ST59-MRSA strains isolated from different hospitals in Shanghai, China, and inhibition assays showed that JD007 could inhibit S. aureus growth at a multiplicity of infection of 0.1. CONCLUSIONS: The results suggested that Staphylococcus phage JD007 can potentially be used in phage therapy or for the detection of S. aureus.
Asunto(s)
Genoma Viral , Especificidad del Huésped , Myoviridae/genética , Myoviridae/fisiología , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/fisiología , Staphylococcus aureus/virología , China , Infección Hospitalaria/microbiología , ADN Viral/química , ADN Viral/genética , Humanos , Concentración de Iones de Hidrógeno , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/efectos de la radiación , Microscopía Electrónica de Transmisión , Myoviridae/clasificación , Myoviridae/aislamiento & purificación , Análisis de Secuencia de ADN , Infecciones Estafilocócicas/microbiología , Fagos de Staphylococcus/clasificación , Fagos de Staphylococcus/aislamiento & purificación , Staphylococcus aureus/aislamiento & purificación , Temperatura , Virión/ultraestructuraRESUMEN
INTRODUCTION: The use of bacteriophages as an alternative treatment method against multidrug-resistant bacteria has not been explored in Kenya. This study sought to determine the efficacy of environmentally obtained lytic bacteriophage against multidrug-resistant Staphylococcus aureus (MDRSA) bacterium in mice. METHODOLOGY: Staphylococcus aureus bacterium and S. aureus-specific lytic phage were isolated from sewage and wastewater collected within Nairobi County, Kenya. Thirty mice were randomly assigned into three groups: MDRSA infection group (n = 20), phage-infection group (n = 5), and non-infection group (n = 5). The MDRSA infection group was further subdivided into three groups: clindamycin treatment (8 mg/kg; n = 5), lytic phage treatment (108 PFU/mL (n = 5), and a combination treatment of clindamycin and lytic phage (n = 5). Treatments were done at either 24 or 72 hours post-infection (p.i), and data on efficacy, bacterial load, and animal physical health were collected. RESULTS: Treatment with phage was more effective (100%) than with clindamycin (62.25% at 24 hours p.i and 87.5% at 72 hours p.i.) or combination treatment (75% at 24 hours p.i. and 90% at 72 hours p.i.) (p < 0.001). CONCLUSIONS: The results show that the environmentally obtained S. aureus lytic bacteriophage has therapeutic potential against MDRSA bacterium in mice.
Asunto(s)
Terapia Biológica/métodos , Infecciones Estafilocócicas/terapia , Fagos de Staphylococcus/crecimiento & desarrollo , Staphylococcus aureus/virología , Animales , Antibacterianos/administración & dosificación , Carga Bacteriana , Clindamicina/administración & dosificación , Terapia Combinada , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana Múltiple , Femenino , Kenia , Masculino , Ratones Endogámicos BALB C , Infecciones Estafilocócicas/patología , Fagos de Staphylococcus/aislamiento & purificación , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Análisis de Supervivencia , Resultado del Tratamiento , Carga Viral , Aguas Residuales/microbiología , Aguas Residuales/virologíaRESUMEN
Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-ß-lactamase enzymes that degrade ß-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-ß-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a ß-lactam antibiotic susceptibility test, the transductant was more highly resistant to ß-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of ß-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.