Combining GC-MS/MS with a LLE-SPE sample pretreatment step to simultaneously analyse enrofloxacin and ofloxacin residues in chicken tissues and pork.

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% âˆ¼ 90.56% enrofloxacin and 78.43% âˆ¼ 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.

Application of a novel lytic phage vB_EcoM_SQ17 for the biocontrol of Enterohemorrhagic Escherichia coli O157:H7 and Enterotoxigenic E. coli in food matrices.

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.

Dysbiosis and intestinal inflammation caused by Salmonella Typhimurium in mice can be alleviated by preadministration of a lytic phage.

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.

The Broad Host Range Phage vB_CpeS_BG3P Is Able to Inhibit Clostridium perfringens Growth.

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.

Virulent phage vB_CpeP_HN02 inhibits Clostridium perfringens on the surface of the chicken meat.

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.

Antimicrobial susceptibility, multilocus sequence typing, and virulence of listeria isolated from a slaughterhouse in Jiangsu, China.

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.

Cellular microRNAs influence replication of H3N2 canine influenza virus in infected cells.

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.

A Spontaneous rapZ Mutant Impairs Infectivity of Lytic Bacteriophage vB_EcoM_JS09 against Enterotoxigenic Escherichia coli.

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.

Biodiversity of New Lytic Bacteriophages Infecting Shigella spp. in Freshwater Environment.

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.

Evidence for the Rapid and Divergent Evolution of Mycoplasmas: Structural and Phylogenetic Analysis of Enolases.

Mycoplasmas are a group of prokaryotes without cell walls that have evolved through several rounds of degenerative evolution. With a low cell DNA G + C content and definitively long genetic lineages, mycoplasmas are thought to be in a state of rapid evolution. However, little associated evidence has been provided. Enolase is a key enzyme in glycolysis that is widely found in all species from the three domains, and it is evolutionarily conserved. In our previous studies, enolase acted as a virulence factor and participated in cell-surface adhesion in Mycoplasma hyopneumoniae. Furthermore, unique loop regions were first found in the crystal structure of Mhp Eno. Here, enolase structures from Mycoplasma pneumoniae and Mycoplasma bovis were determined. An extra helix 7 is specific and conservatively found in almost all mycoplasma enolases, as confirmed by crystal structures and sequence alignment. Particular motifs for helix 7, which is composed of F-K/G-K-L/F-K-X-A-I, have been proposed and could be regarded as molecular markers. To our surprise, the genetic distances between any two mycoplasma enolases were obviously longer than those between the two corresponding species themselves, indicating divergent evolution of mycoplasma enolases, whereas no horizontal gene transfer was detected in mycoplasma enolase genens. Furthermore, different evolutionary patterns were adopted by different loop regions of mycoplasma enolase. Enolases from different Mycoplasma species also showed different affinities for PLG and fibronectin. Our results indicate the rapid and divergent evolution of mycoplasma enolase and mycoplasmas. This study will also aid understanding the independent evolution of Mycoplasma species after separation from their common ancestor.

The complete genome of lytic Salmonella phage vB_SenM-PA13076 and therapeutic potency in the treatment of lethal Salmonella Enteritidis infections in mice.

S. Enteritidis continues to be the most common pathogen of farm animals and a major public health burden worldwide. Using bacteriophages is a potential alternative to antibiotics against S. Enteritidis infection. In this study, the genome analysis of the lytic phage vB_SenM-PA13076 (PA13076) infecting S. Enteritidis revealed a linear, double-stranded DNA genome, which comprised of 52,474 bp and contained 69 ORFs. It belongs to the order Caudovirales; family Myoviridae, genus unclassified. The genes coded for DNA packaging, phage structural proteins, lysis components, DNA recombination, regulation, modification, and replication. No bacterial virulence or drug-resistance genes were detected. The phage PA13076 protected mice from a lethal dose of S. Enteritidis 13076Amp (5 × 108 CFU) by reducing the concentration of bacterial cells in blood, intestine, liver, spleen, and kidney. The phage PA13076 achieved at least 2.5 log reductions of S. Enteritidis cells in infected mice within 24 h (P < 0.05) when compared to the organs of control mice. The data also indicated that phage PA13076 could rapidly enter the blood and four organs of infected mice, remaining therein at concentrations of>104 PFU/g for at least 72 h. These results show that phage PA13076 has definite potential as an antibacterial therapeutic agent for attenuating S. Enteritidis infections.

Emergence of plasmid-mediated high-level tigecycline resistance genes in animals and humans.

Tigecycline is a last-resort antibiotic that is used to treat severe infections caused by extensively drug-resistant bacteria. tet(X) has been shown to encode a flavin-dependent monooxygenase that modifies tigecycline1,2. Here, we report two unique mobile tigecycline-resistance genes, tet(X3) and tet(X4), in numerous Enterobacteriaceae and Acinetobacter that were isolated from animals, meat for consumption and humans. Tet(X3) and Tet(X4) inactivate all tetracyclines, including tigecycline and the newly FDA-approved eravacycline and omadacycline. Both tet(X3) and tet(X4) increase (by 64-128-fold) the tigecycline minimal inhibitory concentration values for Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. In addition, both Tet(X3) (A. baumannii) and Tet(X4) (E. coli) significantly compromise tigecycline in in vivo infection models. Both tet(X3) and tet(X4) are adjacent to insertion sequence ISVsa3 on their respective conjugative plasmids and confer a mild fitness cost (relative fitness of >0.704). Database mining and retrospective screening analyses confirm that tet(X3) and tet(X4) are globally present in clinical bacteria-even in the same bacteria as blaNDM-1, resulting in resistance to both tigecycline and carbapenems. Our findings suggest that both the surveillance of tet(X) variants in clinical and animal sectors and the use of tetracyclines in food production require urgent global attention.

Roles of three TonB systems in the iron utilization and virulence of the Aeromonas hydrophila Chinese epidemic strain NJ-35.

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.

Isolation, characterization, and PCR-based molecular identification of a siphoviridae phage infecting Shigella dysenteriae.

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 62 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.

Development and comparison of liquid-liquid extraction and accelerated solvent extraction methods for quantitative analysis of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in poultry eggs.

Accelerated solvent extraction was investigated as a novel alternative technology for the separation and quantitative analysis of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine from poultry eggs, and the results were compared with the results of liquid-liquid extraction. Rapid quantification of the target compounds was carried out by ultra-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry. This optimized method was validated according to the requirements defined by the European Union and the United States Food and Drug Administration. Finally, the new approach was successfully applied to the quantitative determination of these analytes in 90 commercial poultry eggs from local supermarkets.

Insights Into the Bovine Milk Microbiota in Dairy Farms With Different Incidence Rates of Subclinical Mastitis.

Bovine mastitis continues to be a complex disease associated with significant economic loss in dairy industries worldwide. The incidence rate of subclinical mastitis (IRSCM) can show substantial variation among different farms; however, the milk microbiota, which have a direct influence on bovine mammary gland health, have never been associated with the IRSCM. Here, we aimed to use high-throughput DNA sequencing to describe the milk microbiota from two dairy farms with different IRSCMs and to identify the predominant mastitis pathogens along with commensal or potential beneficial bacteria. Our study showed that Klebsiella, Escherichia-Shigella, and Streptococcus were the mastitis-causing pathogens in farm A (with a lower IRSCM), while Streptococcus and Corynebacterium were the mastitis-causing pathogens in farm B (with a higher IRSCM). The relative abundance of all pathogens in farm B (22.12%) was higher than that in farm A (9.82%). However, the genus Bacillus was more prevalent in farm A. These results may be helpful for explaining the lower IRSCM in farm A. Additionally, the gut-associated genera Prevotella, Ruminococcus, Bacteroides, Rikenella, and Alistipes were prevalent in all milk samples, suggesting gut bacteria can be one of the predominant microbial contamination in milk. Moreover, Listeria monocytogenes (a foodborne pathogen) was found to be prevalent in farm A, even though it had a lower IRSCM. Overall, our study showed complex diversity between the milk microbiota in dairy farms with different IRSCMs. This suggests that variation in IRSCMs may not only be determined by the heterogeneity and prevalence of mastitis-causing pathogens but also be associated with potential beneficial bacteria. In the future, milk microbiota should be considered in bovine mammary gland health management. This would be helpful for both the establishment of a targeted mastitis control system and the control of the safety and quality of dairy products.

Corrigendum: Staphylococcus aureus Bacteriophage Suppresses LPS-Induced Inflammation in MAC-T Bovine Mammary Epithelial Cells.

[This corrects the article DOI: 10.3389/fmicb.2018.01614.].

Alterations in the diversity and composition of mice gut microbiota by lytic or temperate gut phage treatment.

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.

Staphylococcus aureus Bacteriophage Suppresses LPS-Induced Inflammation in MAC-T Bovine Mammary Epithelial Cells.

Several previous studies have shown that bacteriophages can significantly affect the production of various cytokines. The aim of this present study was to investigate the inflammatory effects and mechanisms of bacteriophage vB_SauM_JS25 in stimulated MAC-T bovine mammary epithelial cells by real-time polymerase chain reaction (PCR) and Western blotting. Experiments show that vB_SauM_JS25 reduces Staphylococcus aureus- or lipopolysaccharide (LPS)-induced levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, IL-10, and regulated on activation, normal T cell expressed and secreted (RANTES) mRNA in MAC-T cells, in a manner expected to be unrelated to its antibacterial action. Moreover, S. aureus bacteriophage vB_SauM_JS25 suppressed the LPS-induced phosphorylation of nuclear factor (NF)-κB p65, which may represent an important mechanism mediating these effects. A carefully regulated balance between activation and inhibition by bacteriophages must be kept avoiding inappropriate inflammatory responses. The ability of vB_SauM_JS25 to influence the immune response highlights the potential development and application of bacteriophage-based therapies and may represent a novel anti-inflammatory therapeutic strategy.