RESUMO
BACKGROUND: Listeria monocytogenes is one of the deadliest foodborne pathogens. The bacterium can tolerate severe environments through biofilm formation and antimicrobial resistance. This study aimed to investigate the antimicrobial susceptibility, resistance genes, virulence, and molecular epidemiology about Listeria from meat processing environments. METHODS: This study evaluated the antibiotic resistance and virulence of Listeria isolates from slaughtering and processing plants. All isolates were subjected to antimicrobial susceptibility testing using a standard microbroth dilution method. The harboring of resistant genes was identified by polymerase chain reaction. The multilocus sequence typing was used to determine the subtyping of the isolates and characterize possible routes of contamination from meat processing environments. The virulence of different STs of L. monocytogenes isolates was evaluated using a Caco-2 cell invasion assay. RESULTS: A total of 59 Listeria isolates were identified from 320 samples, including 37 L. monocytogenes isolates (62.71%). This study evaluated the virulence of L. monocytogenes and the antibiotic resistance of Listeria isolates from slaughtering and processing plants. The susceptibility of these 59 isolates against 8 antibiotics was analyzed, and the resistance levels to ceftazidime, ciprofloxacin, and lincomycin were as high as 98.31% (L. m 37; L. innocua 7; L. welshimeri 14), 96.61% (L. m 36; L. innocua 7; L. welshimeri 14), and 93.22% (L. m 35; L. innocua 7; L. welshimeri 13), respectively. More than 90% of the isolates were resistant to three to six antibiotics, indicating that Listeria isolated from meat processing environments had high antimicrobial resistance. Up to 60% of the isolates harbored the tetracycline-resistance genes tetA and tetM. The frequency of ermA, ermB, ermC, and aac(6')-Ib was 16.95, 13.56, 15.25, and 6.78%, respectively. Notably, the resistant phenotype and genotype did not match exactly, suggesting that the mechanisms of antibiotic resistance of these isolates were likely related to the processing environment. Multilocus sequence typing (MLST) revealed that 59 Listeria isolates were grouped into 10 sequence types (STs). The dominant L. monocytogenes STs were ST5, ST9, and ST121 in the slaughtering and processing plant of Jiangsu province. Moreover, ST5 subtypes exhibited high invasion in Caco-2 cells compared with ST9 and ST121 cells. CONCLUSION: The dominant L. monocytogenes ST5 persisted in the slaughtering and processing plant and had high antimicrobial resistance and invasion characteristics, illustrating a potential risk in food safety and human health.
Assuntos
Antibacterianos/farmacologia , Listeria/efeitos dos fármacos , Listeria/patogenicidade , Matadouros/estatística & dados numéricos , Animais , China , Farmacorresistência Bacteriana , Inocuidade dos Alimentos , Humanos , Listeria/classificação , Listeria/genética , Carne/microbiologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Suínos/microbiologia , VirulênciaRESUMO
BACKGROUND: Shigella dysenteriae is one of the members of Shigella genus which was the main responsible of different Shigellosis outbreaks worldwide. The increasing consumption of antibiotics has led to the emergence and spreading of antibiotic-resistant strains. Therefore, finding new alternatives for infection control is essential, one of which is using bacteriophages. MATERIALS AND METHODS: Lytic bacteriophage against Shigella dysenteriae was isolated from petroleum refinery wastewater. Phage morphological and genetic characteristics were studied using TEM, and sequencing, respectively. In addition, the genome size was estimated, and phage resistance to different temperatures and pH, host range, adsorption rate, and one-step growth were investigated. RESULTS: According to the morphology and genetic results, this phage was named vB-SdyS-ISF003. Sequencing of the PCR products revealed that the vB-SdyS-ISF003 phage belongs to the species T1virus, subfamily Tunavirinae of family Siphoviridae. This was the first detected bacteriophage against S. dysenteriae, which belongs to the family Siphoviridae. In addition, its host range was limited to S. dysenteriae. The genome size was about 62â¯kb. vB-SdyS-ISF003 phage has a number of desirable characteristics including the limited host range to S. dysenteriae, very short connection time, a relatively wide range of temperature tolerance -20 to 50⯰C, pH tolerance of 7-9 without significant reduction in the phage titer. CONCLUSION: vB-SdyS-ISF003 is a novel virulent T1virus phage and has the appropriate potential for being used in bio controlling of S. dysenteriae in different condition.
Assuntos
Reação em Cadeia da Polimerase/métodos , Shigella dysenteriae/virologia , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/isolamento & purificação , DNA Viral/genética , Tamanho do Genoma , Genoma Viral , Especificidade de Hospedeiro , Concentração de Íons de Hidrogênio , Terapia por Fagos , Shigella dysenteriae/patogenicidade , Siphoviridae/crescimento & desenvolvimento , Temperatura , TermotolerânciaRESUMO
The TonB system functions in iron transport and has been identified in certain Gram-negative bacteria. Recently, we reported three TonB systems in the Aeromonas hydrophila Chinese epidemic strain NJ-35, but the functions of these systems have not been thoroughly elucidated to date. In this study, we investigated the role of these TonB systems in A. hydrophila iron utilization and virulence. We found that tonB1 and tonB2 were preferentially transcribed in iron-chelated conditions, where gene expression levels were approximately 8- and 68-fold higher compared with iron-rich conditions, respectively; tonB3 was consistently transcribed at a low level under iron-repleted and iron-depleted conditions. Only the TonB2 system was required to utilize iron-binding proteins. The tonB123 mutant showed increased susceptibility to erythromycin and roxithromycin. In addition, all three tonB genes were involved in A. hydrophila virulence in zebrafish, and various phenotypes associated with environmental survival were changed with varying degrees in each tonB mutant. TonB2 plays a relatively major role in adhesion, motility, and biofilm formation, while TonB3 is more involved in the anti-phagocytosis of A. hydrophila. In each observed phenotype, no significant difference was found between the single- and double-deletion mutants, whereas the triple-deletion mutant exhibited the most serious defects, indicating that all three TonB systems of A. hydrophila coordinately complement one another. In conclusion, this study elucidates the importance of TonB in iron acquisition and virulence of A. hydrophila, which lays the foundation for future studies regarding the survival mechanisms of this bacterium in iron-restricted environments.
Assuntos
Aeromonas hydrophila/isolamento & purificação , Aeromonas hydrophila/patogenicidade , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Virulência/metabolismo , Animais , Aquicultura , Proteínas de Bactérias/genética , China , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Proteínas de Membrana/genética , Análise de Sobrevida , Oligoelementos/metabolismo , Virulência , Fatores de Virulência/genética , Peixe-ZebraRESUMO
Primary porcine bronchial epithelial cells (PBECs) are an ideal model to study the molecular and pathogenic mechanisms of various porcine respiratory pathogens. However, the short lifespan of primary PBECs greatly limit their application. Here, we isolated and cultured primary PBECs and established immortalized PBECs by transfecting primary PBECs with the pEGFP-hTERT recombinant plasmid containing human telomerase reverse transcriptase (hTERT). Immortalized PBECs (hTERT-PBECs) retained the morphological and functional features of primary PBECs as indicated by cytokeratin 18 expression, telomerase activity assay, proliferation assays, karyotype analysis, and quantitative reverse-transcriptase polymerase chain reaction. Compared to primary PBECs, hTERT-PBECs had higher telomerase activity, extended replicative lifespan, and displayed enhanced proliferative activity. Moreover, this cell line is not transformed in vitro and does not exhibit a malignant phenotype in vivo, suggesting that it can be safely used in further studies. Besides, hTERT-PBECs were susceptible to swine influenza virus of H3N2 subtype and porcine circovirus type 2. In conclusion, the immortalized hTERT-PBECs represent a valuable in vitro model, which can be widely used in the study of porcine respiratory pathogenic infections.
Assuntos
Brônquios/citologia , Células Epiteliais/enzimologia , Cultura Primária de Células/métodos , Telomerase/genética , Animais , Brônquios/enzimologia , Proliferação de Células/genética , Circovirus/patogenicidade , Humanos , Cariótipo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , Suínos , Telomerase/biossínteseRESUMO
The type VI secretion system (T6SS) has been considered as a crucial factor in bacterial competition and virulence. The hemolysin co-regulated protein (Hcp) is the hallmark of T6SS. The secretion of Hcp in Aeromonas hydrophila Chinese epidemic strain NJ-35 indicated a functional T6SS. In this study, three copies of the hcp gene were identified in the genome of strain NJ-35. We targeted these Hcp family proteins for generating deletion mutants. These mutants showed varying levels in Hcp production, the interaction with other bacteria or eukaryotic cells, and bacterial virulence. Hcp1 was necessary for T6SS assembly and played a predominant role in the bacterial competition; Hcp2 negatively functioned in the biofilm formation and bacterial adhesion and was more involved in the A. hydrophila virulence in zebrafish and survival against the predation of Tetrahymena, and Hcp3 positively influenced the biofilm formation and bacterial adhesion. These findings illustrate that the T6SS of A. hydrophila NJ-35 is active, and the three Hcp family proteins take part in different processes in environmental adaptation and virulence of this bacterium. This study will provide valuable insights into our understanding of microbial interactions and thus contribute to a broader effort to manipulate these interactions for therapeutic or environmental benefit.
Assuntos
Aeromonas hydrophila/metabolismo , Aeromonas hydrophila/patogenicidade , Proteínas de Bactérias/genética , Proteínas Hemolisinas/genética , Virulência/genética , Aeromonas hydrophila/genética , Animais , Aderência Bacteriana/genética , Biofilmes , China , Microbiologia Ambiental , Deleção de SequênciaRESUMO
Phages, the most abundant species in the mammalian gut, have numerous advantages as biocontrol agent over antibiotics. In this study, mice were orally treated with the lytic gut phage PA13076 (group B), the temperate phage BP96115 (group C), no phage (group A), or streptomycin (group D) over 31 days. At the end of the experiment, fecal microbiota diversity and composition was determined and compared using high-throughput sequencing of the V3-V4 hyper-variable region of the 16S rRNA gene and virus-like particles (VLPs) were quantified in feces. There was high diversity and richness of microbiota in the lytic and temperate gut phage-treated mice, with the lytic gut phage causing an increased alpha diversity based on the Chao1 index (p < 0.01). However, the streptomycin treatment reduced the microbiota diversity and richness (p = 0.0299). Both phage and streptomycin treatments reduced the abundance of Bacteroidetes at the phylum level (p < 0.01) and increased the abundance of the phylum Firmicutes. Interestingly, two beneficial genera, Lactobacillus and Bifidobacterium, were enhanced by treatment with the lytic and temperate gut phage. The abundance of the genus Escherichia/Shigella was higher in mice after temperate phage administration than in the control group (p < 0.01), but lower than in the streptomycin group. Moreover, streptomycin treatment increased the abundance of the genera Klebsiella and Escherichia/Shigella (p < 0.01). In terms of the gut virome, fecal VLPs did not change significantly after phage treatment. This study showed that lytic and temperate gut phage treatment modulated the composition and diversity of gut microbiota and the lytic gut phage promoted a beneficial gut ecosystem, while the temperate phage may promote conditions enabling diseases to occur.
Assuntos
Bacteriófagos/fisiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Animais , Bacteriólise , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/virologia , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/virologia , Escherichia/efeitos dos fármacos , Escherichia/virologia , Fezes/microbiologia , Feminino , Firmicutes/efeitos dos fármacos , Firmicutes/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Klebsiella/efeitos dos fármacos , Klebsiella/virologia , Lactobacillus/efeitos dos fármacos , Lactobacillus/virologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Shigella/efeitos dos fármacos , Shigella/virologia , Estreptomicina/farmacologiaRESUMO
BACKGROUND: Avian-origin H3N2 canine influenza virus (CIV) has been the most common subtype in Korea and China since 2007. Here, we compared the pathogenicity and transmissibility of three H3N2 CIV strains [Chinese CIV (JS/10), Korean CIV (KR/07), and Korean recombinant CIV between the classic H3N2 CIV and the pandemic H1N1 virus (MV/12)] in BALB/c mouse and guinea pig models. The pandemic H1N1 (CA/09) strain served as the control. RESULTS: BALB/c mice infected with H1N1 had high mortality and obvious body weight loss, whereas no overt disease symptoms were observed in mice inoculated with H3N2 CIV strains. The viral titers were higher in the group MV/12 than those in groups JS/10 and KR/07, while the mice infected with JS/10 showed higher viral titers in all tissues (except for the lung) than the mice infected with KR/07. The data obtained in guinea pigs also demonstrated that group MV/12 presented the highest loads in most of the tissues, followed by group JS/10 and KR/07. Also, direct contact transmissions of all the three CIV strains could be observed in guinea pigs, and for the inoculated and the contact groups, the viral titer of group MV/12 and KR/07 was higher than that of group JS/10 in nasal swabs. These findings indicated that the matrix (M) gene obtained from the pandemic H1N1 may enhance viral replication of classic H3N2 CIV; JS/10 has stronger viral replication ability in tissues as compared to KR/07, whereas KR/07 infected guinea pigs have more viral shedding than JS/10 infected guinea pigs. CONCLUSIONS: There exists a discrepancy in pathobiology among CIV isolates. Reverse genetics regarding the genomes of CIV isolates will be helpful to further explain the virus characteristics.
Assuntos
Doenças do Cão/virologia , Vírus da Influenza A Subtipo H3N2/patogenicidade , Infecções por Orthomyxoviridae/veterinária , Animais , China/epidemiologia , Modelos Animais de Doenças , Cães , Feminino , Cobaias , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , República da Coreia/epidemiologia , Carga Viral , VirulênciaRESUMO
OBJECTIVES: To investigate the epidemiological characteristics and the surrounding genetic structure of the blaNDM-5 gene in Klebsiella pneumoniae derived from dairy cows in Jiangsu Province, China. METHODS: Ten carbapenem-resistant K. pneumoniae were collected from three dairy farms and were screened for the presence of carbapenemase genes using PCR and sequencing. PFGE and MLST were conducted to analyse the genetic relatedness of the blaNDM-5-harbouring K. pneumoniae isolates. The location of blaNDM-5 was identified by S1 nuclease-PFGE and Southern blotting. The transferability and profiles of blaNDM-5-carrying plasmids were analysed by conjugation experiments, and PCR-based replicon typing and RFLP, respectively. The surrounding genetic structure of the blaNDM-5 gene was obtained using WGS and PCR mapping. RESULTS: All 10 K. pneumoniae from dairy cows harboured the blaNDM-5 gene, exhibited resistance to multiple antimicrobials and belonged to five STs, of which ST1661 and ST2108 were the most prevalent. The blaNDM-5 gene was located on the â¼46 kb IncX3 plasmid in all isolates and these plasmids could be conjugated to an Escherichia coli recipient with no additional resistance profiles transferred. All blaNDM-5-carrying plasmids shared similar genetic contexts and were nearly identical to that of the human K. pneumoniae plasmid (pNDM-MGR194) previously reported in India. CONCLUSIONS: This was the first case of blaNDM-5-positive K. pneumoniae identified from dairy cows in China. The IncX3 pNDM-MGR194-like plasmid disseminated among cow farms should be highlighted and its potential role in mediating transmission of the blaNDM gene between bacteria from humans and animals requires further monitoring.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Klebsiella/veterinária , Klebsiella pneumoniae/isolamento & purificação , beta-Lactamases/análise , Animais , Southern Blotting , Bovinos , China , Conjugação Genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Ordem dos Genes , Transferência Genética Horizontal , Genótipo , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/genética , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , beta-Lactamases/genéticaRESUMO
Bovine mastitis caused by Streptococcus agalactiae continues to be one of the major veterinary and economic issues in certain areas of the world. The more prevalent S. agalactiae strains that cause bovine mastitis in China dairy farms belong to a number of bovine-adapted sequence types (STs) ST67, ST103 and ST568. However, it is unknown why these STs can emerge as highly prevalent clones in bovine dairy farms. Here, to determine if a variety of virulence characteristics were associated with these highly prevalent STs, the molecular and virulence characterization of 116 strains isolated from bovine, human, fish and environment were analyzed. Our data showed that all bovine-adapted strains could be assigned to capsular genotype Ia or II, and carried pilus island 2b, and lactose operon. Importantly, we demonstrated that the growth ability in milk, biofilm formation ability and adhesion ability to bovine mammary epithelial cells (BMECs) were significantly higher for all bovine-adapted strains compared to strains from other origins. Additionally, ST103 and ST568 strains exhibited significantly higher hemolytic activity and cytotoxicity than ST67 strains. In conclusion, our study provides substantial evidence for the hypothesis that the virulence characteristics including efficient growth in milk, elevated biofilm formation ability, together with strong adhesion ability might have favored the high prevalence of the STs in the bovine environment, whereas the hemolytic activity and cytotoxicity were not the crucial characteristics.
Assuntos
Mastite Bovina/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/fisiologia , Streptococcus agalactiae/patogenicidade , Animais , Bovinos , China , Indústria de Laticínios , Feminino , Genótipo , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , VirulênciaRESUMO
Canine influenza virus (CIV) subtype H3N2 is a newly identified, highly contagious respiratory pathogen that causes cough, pneumonia and other respiratory symptoms in dogs. Data indicate that the virus is responsible for recent clinical cases of dog disease in China. However, therapeutic options for this disease are very limited. In this study, seven monoclonal antibodies (mAbs) against CIV JS/10 (an H3N2 subtype virus) were produced and characterized. Among them, mAb D7, which is specific for the HA2 glycopeptide (gp), induced the highest neutralization titers. The protection provided by mAb D7 was evaluated in BALB/c mice challenged with homologous or heterologous strains of H3N2 influenza virus, including two strains of CIV and one strain of swine influenza virus (SIV). The data show that mAb D7 protected the mice from infection with the three viral strains, especially the homologous strain, which was indicated by the recovery of body weight, reduction of viral load, and reduction of tissue damage. Moreover, the levels of IFN-γ and TNF-α in the lungs, as detected by ELISA, were reduced in the infected mice treated with the mAb D7 compared with those without mAb D7 treatment. Thus, our findings demonstrate, for the first time, that a mAb could reduce the release of IFN-γ and TNF-α associated with tissue damage by CIV infection and that the mAb might be of great therapeutic value for CIV infection.
Assuntos
Anticorpos Antivirais/imunologia , Doenças do Cão/imunologia , Glicopeptídeos/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Infecções por Orthomyxoviridae/veterinária , Proteínas Virais/genética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Doenças do Cão/virologia , Cães , Feminino , Glicopeptídeos/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Análise de Sequência de DNA , Proteínas Virais/metabolismoRESUMO
Motile aeromonad septicemia (MAS) caused by Aeromonas hydrophila is one of the common bacterial causes of fish mortalities. Prophylactic vaccination against this and other diseases is essential for continued growth of aquaculture. The type VI secretion system (T6SS) plays a crucial role in the virulence of A. hydrophila. The hemolysin co-regulated protein (Hcp) is an integral component of the T6SS apparatus and is considered a hallmark of T6SS function. Here, the T6SS effector Hcp was expressed and characterized, and its immunogenicity and protective efficacy were evaluated in common carp (Cyprinus carpio). Hcp secretion was found to be strongly induced by low temperature in A. hydrophila. Immunoblot analysis demonstrated that Hcp is conserved among A. hydrophila strains of different origins. The vaccination with recombinant Hcp resulted in an increased survival (46.67%) in common carp during a 10-day challenge time compared to non-vaccinated fish (7.14%). The vaccinated fish also showed the significantly increased levels of IgM antibody in serum and cytokines such as inerleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in kidney, spleen and gills. The recombinant Hcp shows promise as a vaccine candidate against A. hydrophila.
Assuntos
Aeromonas hydrophila/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Carpas , Doenças dos Peixes/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Proteínas Hemolisinas/imunologia , Aeromonas hydrophila/genética , Animais , Proteínas de Bactérias/genética , Citocinas/genética , Citocinas/metabolismo , Doenças dos Peixes/microbiologia , Genoma Bacteriano , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Proteínas Hemolisinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sequência de DNA/veterináriaRESUMO
Dogs are susceptible to infectious diseases that occur primarily in the respiratory tract. The airway epithelium acts as a first line of defense and is constantly exposed to microorganisms present in the environment. Respiratory epithelial cells have recently gained wide use as a cell model for studying the pathogenesis of human, murine or swine respiratory pathogen infections. However, studies of the pathogenic mechanisms of canine pathogens have been hindered by the lack of reliable respiratory cell lines. Here, we cultured primary canine bronchiolar epithelial cells (CBECs), whose characteristics were confirmed by their expression of the epithelial cell-specific marker cytokeratin 18, and have provided protocols for their isolation and ex vivo expansion. Further, we established immortalized CBECs containing the human telomerase reverse transcriptase (hTERT) gene via transfection of primary CBECs with the recombinant plasmid pEGFP-hTERT. Immortalized bronchiolar epithelial cells (hTERT-CBECs) retain the morphological and functional features of primary CBECs, as indicated by reverse transcriptase polymerase chain reaction, proliferation assays, karyotype analysis, telomerase activity assay, and Western blotting, which demonstrate that hTERT-CBECs have higher telomerase activity, an extended proliferative lifespan, and a diploid complement of chromosomes, even after Passage 50. Moreover, this cell line is not transformed, as evaluated using soft agar assays and tumorigenicity analysis in nude mice, and can therefore be safely used in future studies. The isolation and establishment of stable hTERT-CBECs is of great importance for use as an in vitro model for mechanistic studies of canine pathogenic infections.
Assuntos
Linhagem Celular , Células Epiteliais/fisiologia , Telomerase/genética , Telomerase/metabolismo , Animais , Proliferação de Células , Diploide , Cães , Células Epiteliais/enzimologia , Expressão Gênica , Instabilidade Genômica , Humanos , Camundongos , Camundongos Nus , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
A widely applicable original gas chromatography-tandem mass spectrometry (GC-MS/MS) method was explored to qualitatively and quantitatively measure enrofloxacin and ofloxacin residues in chicken tissues and pork. The experimental samples were processed based on liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Trimethylsilyl diazomethane (TMSD) was chosen to react derivatively with enrofloxacin and ofloxacin. In total, 78.25% â¼ 90.56% enrofloxacin and 78.43% â¼ 91.86% ofloxacin was recovered from the blank fortified samples. The limits of detection (LODs) were 0.7-1.0 µg/kg and 0.1-0.2 µg/kg, respectively. The limits of quantitation (LOQs) were 1.6-1.9 µg/kg and 0.3-0.4 µg/kg, respectively. It was verified that various experimental data met the requirements of the FAO & WHO (2014) for the detection of veterinary drug residues. Real samples obtained from local markets were analysed using the established method, and no residues of enrofloxacin or ofloxacin were detected in the samples.
Assuntos
Antibacterianos , Galinhas , Resíduos de Drogas , Enrofloxacina , Contaminação de Alimentos , Cromatografia Gasosa-Espectrometria de Massas , Carne , Ofloxacino , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Animais , Enrofloxacina/análise , Resíduos de Drogas/análise , Resíduos de Drogas/química , Suínos , Extração em Fase Sólida/métodos , Contaminação de Alimentos/análise , Carne/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Ofloxacino/análise , Antibacterianos/análise , Extração Líquido-Líquido/métodos , Fluoroquinolonas/análiseRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and Enterotoxigenic E. coli (ETEC) are important foodborne pathogens, causing serious food poisoning outbreaks worldwide. Bacteriophages, as novel antibacterial agents, have been increasingly exploited to control foodborne pathogens. In this study, a novel broad-host range lytic phage vB_EcoM_SQ17 (SQ17), was isolated, characterized, and evaluated for its potential to control bacterial counts in vitro and in three different food matrices (milk, raw beef, and fresh lettuce). Phage SQ17 was capable of infecting EHEC O157:H7, ETEC, and other E. coli strains. Morphology, one-step growth, and stability assay showed that phage SQ17 belongs to the Caudovirales order, Myoviridae family, and Mosigvirus genus. It has a short latent period of 10 min, a burst size of 71 PFU/infected cell, high stability between pH 4 to 12 as well as thermostability between 30°C and 60°C for 60 min. Genome sequencing analysis revealed that the genome of SQ17 does not contain any genes associated with antibiotic resistance, toxins, lysogeny, or virulence factors, indicating the potential safe application of phage SQ17 in the food industry. In Luria-Bertani (LB) medium, phage SQ17 significantly decreased the viable counts of EHEC O157:H7 by more than 2.40 log CFU/ml (p < 0.05) after 6 h of incubation at 37°C. Phage SQ17 showed great potential to be applied for biocontrol of EHEC O157:H7 in milk and raw beef. In fresh lettuce, treatment with SQ17 also resulted in significant reduction of viable cell counts of EHEC O157:H7 and ETEC at both 4°C and 25°C. Our results demonstrate that SQ17 is a good candidate for application as an EHEC O157:H7 and ETEC biocontrol agent in the processing stages of food production and food preservation.
RESUMO
Clostridium perfringens is a well-known pathogen that causes foodborne disease. With a high prevalence of contamination in food, an efficient strategy is needed to decontaminate those contaminated foods and control the emergence of foodborne disease. In this study, the C. perfringens-specific lytic phage vB_CpeP_HN02 (designated as phage HN02) was isolated from chicken feces. Electron microscopy and phylogenetic analysis suggested that phage vB_CpeP_HN02 is a novel phage of the family Podoviridae. Phage HN02 had good pH (5-11) and temperature tolerance (< 70 °C). Phage HN02 exhibited a broad host range of C. perfringens isolates (42.86%). The complete genome of the phage HN02 was sequenced and revealed a linear double-stranded DNA genome. The 17,754-bp genome (GenBank MW815121) with average GC content of 28.2% includes 22 predicted open reading frames, of which only 10 were annotated with known functions. Phylogenetic analysis of the available C. perfringens phage major capsid protein demonstrated that phage HN02 is closely related to virulent C. perfringens phage phi24R and CPD2. When phage HN02 was applied to chicken meat samples stored at 4 °C for 72 h with 1 × 106 to 1 × 109 PFU/g, 95% to 99% of C. perfringens were inactivated on chicken meat surfaces after storage at 4 °C for 72 h, respectively. It should be noted that C. perfringens could be completely lysed by a high dose of phage HN02 (1 × 1010 PFU/g) after 48 h treatment in chicken samples. Through the lytic activity testing, phage HN02 showed good antimicrobial effects, and can be used as an antibacterial agent for biocontrol of C. perfringens in meat products.
Assuntos
Bacteriófagos , Animais , Bacteriófagos/genética , Galinhas , Clostridium perfringens , Carne , FilogeniaRESUMO
Clostridium perfringens is an important pathogen for both humans and animals, causing human foodborne disease and necrotic enteritis in poultry. In the present study, a C. perfringens-specific phage, vB_CpeS_BG3P (designated as BG3P hereafter), was isolated from chicken farm sewage. Both electron microscopy and phylogenetic analysis suggested that phage BG3P is a novel phage belonging to Siphoviridae family. Phage BG3P exhibited a broad host range against different C. perfringens isolates (90.63% of strains were infected). Sequencing of the complete genome revealed a linear double-stranded DNA (43,528 bp) with 28.65% GC content. After sequence analysis, 73 open reading frames (orfs) were predicted, of which only 13 were annotated with known functions. No tRNA and virulence encoding genes were detected. It should be noted that the protein of orf 15 has 97.92% homology to C. perfringens-specific chloramphenicol resistance protein, which has not been reported for any C. perfringens phage. Phylogenetic analysis of the ssDNA binding protein demonstrated that this phage is closely related to C. perfringens phages phiSM101 and phi3626. In considering future use as an antimicrobial agent, some biological characteristics were observed, such as a good pH (3−11) stability and moderate temperature tolerance (<60 °C). Moreover, bacteriophage BG3P showed a good antimicrobial effect against C. perfringens liquid cultures. Thus, phage treatment with MOI ≥ 100 completely inhibited bacterial growth compared to untreated cultures. Although phage BG3P shows good lytic efficiency and broad host range in vitro, future development and application may need to consider removal of the chloramphenicol-like resistance gene or exploring its lysin for future antibacterial applications.
Assuntos
Bacteriófagos , Siphoviridae , Animais , Clostridium perfringens/genética , Genoma Viral , Especificidade de Hospedeiro , Filogenia , Siphoviridae/genéticaRESUMO
Many studies have shown the efficacy of phage therapy in reducing intestinal pathogens. However, phage-based probiotic treatment is poorly studied in view of effects on the gut microbiota and intestinal inflammation. In this study, a lytic or a temperate phage (each at 4 ×108 PFU per day) or a streptomycin solution (40 mg per day) were administered to mice via drinking water for 31 days. Subsequently, mice were challenged with Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium). S. Typhimurium does not serve as the host bacterium and is not lysed by both phages. For intestinal inflammation evaluation, mice were given one dose of streptomycin for 24 h before the S. Typhimurium challenge. High-throughput sequencing analysis revealed that the phylum Firmicutes became the most abundant in mice pretreated with phages. The alpha diversity of gut bacteria was higher in phage treated than in streptomycin treated mice. Moreover, pretreatment with the lytic and the temperate phage before the S. Typhimurium challenge increased two beneficial genera, Lactobacillus and Bifidobacterium. According to the pathological analysis of ileum, cecum, and serum, temperate or lytic gut phage pretreatment of mice markedly reduced intestinal inflammation, concomitant with lower serum concentration of lipopolysaccharides (LPS) and diamine oxidase (DAO). The oral pretreatments of mice (ST, Lyt, Lys, SM) generally caused an increased expression of IL-1ß, TNF-α, IFN-γ, IL-4, and IL-10 compared to the matching control. However, in mice pretreated with the lytic phage, the mRNA expression for the pro-inflammatory cytokine TNF-α was not significantly higher than that of the control group. No significant differences were detected for peripheral blood B lymphocytes, CD3 +T cells, and the CD4 + /CD8 + ratio in mice pretreated with the lytic and lysogenic phage. This study demonstrated that even lytic phages not targeting the pathogenic serovar Salmonella Typhimurium alleviated intestinal dysbiosis and inflammation in challenged mice.
Assuntos
Bacteriófagos , Salmonelose Animal , Animais , Disbiose/terapia , Inflamação/terapia , Camundongos , Salmonelose Animal/microbiologia , Salmonella typhimurium , EstreptomicinaRESUMO
Our understanding of the mechanisms underlying phage-bacterium interactions remains limited. In Escherichia coli, RapZ regulates glucosamine-6-phosphate (GlcN6P) metabolism, the formation of which initiates synthesis of the bacterial cell envelope, including lipopolysaccharides (LPS). However, the role of RapZ, if any, on phage infectivity remains to be investigated. Here, we isolated strains of enterotoxigenic E. coli (ETEC) resistant to its specific lytic bacteriophage vB_EcoM_JS09 (JS09) in a phage aerosol spray experiment. Whole-genome analysis of phage-resistant bacteria revealed the rapZ gene acquired a premature stop mutation at amino acid 227. Here, we report that the mutation in the rapZ gene confers resistance by inhibiting 93.5% phage adsorption. Furthermore, this mutation changes the morphology of phage plaques, reduces efficiency of plating and phage propagation efficiency, and impairs the infectivity of phage JS09 against ETEC. Using scanning electron microscopy assays, we attribute the inability of the phage to adsorb to the loss of receptors in strains with defective RapZ. Analysis of the LPS profile shows that strains with defective RapZ inhibit phage infection by changing the LPS profile in E. coli Preincubation of phage JS09 with LPS extracted from a wild-type (WT) strain blocked infection, suggesting LPS is the host receptor for phage JS09 adsorption. Our data uncover the mechanism by which ETEC resists infection of phage JS09 by mutating the rapZ gene and then increasing the expression of glmS and changing the phage receptor-LPS profile. These findings provide insight into the function of the rapZ gene for efficient infection of phage JS09.IMPORTANCE The development of phage-resistant bacteria is a challenging problem for phage therapy. However, our knowledge of phage resistance mechanisms is still limited. RapZ is an RNase adaptor protein encoded by the rapZ gene and plays an important function in Gram-positive and Gram-negative bacteria. Here, we report the whole-genome analysis of a phage-resistant enterotoxigenic Escherichia coli (ETEC) strain, which revealed that the rapZ gene acquired a premature stop mutation (E227Stop). We show that the premature stop mutation of rapZ impairs the infectivity of phage JS09 in ETEC. Furthermore, our findings indicate that ETEC becomes resistant against the adsorption and infection of phage JS09 by mutating the rapZ gene, increasing the expression of glmS, and changing the phage receptor-LPS profile. It is also first reported here that RapZ is essential for efficient infection of phage JS09.
Assuntos
Bacteriófagos/patogenicidade , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/virologia , Proteínas de Escherichia coli/genética , Mutação , Proteínas de Ligação a RNA/genética , Proteínas de Bactérias/genética , Interações entre Hospedeiro e Microrganismos/genética , Ligação ViralRESUMO
MicroRNAs (miRNAs) are known to play important regulatory roles in host-virus interactions. Avian-origin H3N2 canine influenza virus (CIV) has emerged as the most prevalent subtype among dogs in Asia since 2007. To evaluate the roles of host miRNAs in H3N2 CIV infection, here, miRNA profiles obtained from primary canine bronchiolar epithelial cells (CBECs) and canine alveolar macrophages (CAMCs) were compared between infected and mock-infected cells with the H3N2 CIV JS/10. It was found that the expressions of cfa-miR-125b and cfa-miR-151, which have been reported to be associated with innate immunity and inflammatory response, were significantly decreased in CIV-infected canine primary cells. Bioinformatics prediction indicated that 5' seed regions of the two miRNAs are partially complementary to the mRNAs of nucleoprotein (NP) and non-structural protein 1 (NS1) of JS/10. As determined by virus titration, quantitative real-time PCR (qRT-PCR) and western blotting, overexpression of the two miRNAs inhibited CIV replication in cell culture, while their inhibition facilitated this replication, suggesting that the two miRNAs could act as negative regulators of CIV replication. Our findings support the notion that some cellular miRNAs can influence the outcome of virus infection, which helps to elucidate the resistance of host cells to viral infection and to clarify the pathogenesis of H3N2 CIV.
Assuntos
Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Macrófagos Alveolares/virologia , MicroRNAs/genética , Replicação Viral/genética , Animais , Brônquios/citologia , Células Cultivadas , Doenças do Cão/virologia , Cães , Células Epiteliais/virologia , Vírus da Influenza A Subtipo H3N2/genética , Células Madin Darby de Rim Canino , MasculinoRESUMO
Bacteriophages, viruses that infect and replicate within prokaryotic cells are the most abundant life forms in the environment, yet the vast majority of them have not been properly reported or even discovered. Almost all reported bacteriophages infecting the Enterobacteriaceae family, with Escherichia coli being the major subject of studies, have been isolated from wastewater, sewage, and effluent resources. In the present study, we focused on the distribution and biodiversity of Shigella phages in an aquatic ecosystem. While no Shigella bacteria was recovered from the Yangtze River, three lytic phages were isolated from this ecosystem and were subjected to biological, morphological, and genomic characteristics. Comparative genomics and phylogenetic analyses demonstrated that vB _SflM_004 isolate belongs to Myoviridae family, Felixounavirus genus of Ounavirinae subfamily, vB_SdyM_006 was classified under the same family, however, it is suggested to be in a new genus under Tevenvirinae subfamily with some other related bacteriophages. vB_SsoS_008 phage belongs to the Siphoviridae family, Tunavirus genus, Tunavirinae subfamily. The phages did not harbor any genes involved in the lysogenic cycles and showed a high temperature and pH stability. The biodiversity of the isolated phages highly suggests that continued isolation on non-model members of Enterobacteriaceae family is necessary to fully understand bacteriophage diversity in aquatic environments.