Perturbations of gut microbiome and metabolome of pigs infected with Mycoplasma hyorhinis.

BACKGROUND: Mycoplasma hyorhinis is a prevalent respiratory pathogen in swine, causing significant economic loss to pig producers. There is growing evidence that respiratory pathogen infections have a large impact on intestinal microecology. To study the effect of M. hyorhinis infection on gut microbial composition and metabolome profile, pigs were infected with M. hyorhinis. Metagenomic sequencing analysis was performed of fecal samples and a liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of gut digesta was made. RESULTS: Pigs infected with M. hyorhinis had enriched Sutterella and Mailhella, and depleted Dechloromonas, Succinatimonas, Campylobacter, Blastocystis, Treponema, and Megasphaera. The pigs infected with M. hyorhinis also had greater abundances of bacterium_0_1xD8_71, Ruminococcus_sp__CAG_353, Firmicutes_bacterium_CAG_194, Firmicutes_bacterium_CAG_534, bacterium_1xD42_87, and lower abundances of Chlamydia_suis, Megasphaera_elsdenii, Treponema_porcinum, Bacteroides_sp__CAG_1060, Faecalibacterium_prausnitzii. Metabolomic analysis revealed that some lipids and lipid-like molecules increased in the small intestine, whereas most lipids and lipid-like molecule metabolites decreased in the large intestine. These altered metabolites induce changes in intestinal sphingolipid metabolism, amino acid metabolism, and thiamine metabolism. CONCLUSION: These findings demonstrate that infection with M. hyorhinis can alter the gut microbial composition and metabolite structure in pigs, which may further affect amino acid metabolism and lipid metabolism in the intestine. © 2023 Society of Chemical Industry.

Encapsulated Essential Oils Improve the Growth Performance of Meat Ducks by Enhancing Intestinal Morphology, Barrier Function, Antioxidant Capacity and the Cecal Microbiota.

The objective of this study was to evaluate the effects of encapsulated essential oils (EOs) on the gut microbiota, growth performance, intestinal morphology, antioxidant properties and barrier function of meat-type ducks. A total of 320 male Cherry Valley ducks (1 day old), were randomly assigned to four dietary experimental groups with eight replicates of ten ducks each. The groups consisted of the CON group (basal diet), the HEO group (basal diet + EO 1000 mg/kg), the LEO group (basal diet + EO 500 mg/kg), and the ANT group (basal diet + chlortetracycline 50 mg/kg). Our findings indicated that ducks fed with EO 1000 mg/kg had greater average daily feed intake (ADFI), average daily gain (ADG), and body weight (BW) and a lower feed conversion ratio (FCR) than the other groups. The serum concentration of TG reduced in the HEO (p > 0.05) and LEO (p < 0.05) groups on day 42, while the concentration of CHOL increased with the EO concentration in the LEO (p > 0.05) and HEO (p < 0.05) groups. No differences were observed in the ileal mucosa for the activities of SOD, MPO and GSH-PX after EO dietary treatment. Dietary supplementation with EOs significantly increased the villus heights (p < 0.01) and the ratio of villus height to crypt depth (c/v) in the duodenum and jejunum of ducks. Moreover, the mRNA expressions of Claudin1 and Occludin in the jejunal mucosa were observed to be higher in the LEO and HEO groups rather than the CON and ANT groups on d 42. The α diversity showed that the HEO group improved the bacterial diversity and abundance. The ß diversity analysis indicated that the microbial structures of the four groups were obviously separated. EO dietary supplementation could increase the relative abundance (p < 0.01) of the Bacteroidetes phylum, Bacteroidaceae family, and Bacteroides, Desulfovibrio, Phascolarctobacterium, and Butyricimonas genera in the cecal microbiota of ducks. We demonstrated significant differences in the bacterial composition and functional potential of the gut microbiota in ducks that were fed either an EO diet or a basal diet. Therefore, supplemented EOs was found to have a positive effect on the growth performance and intestinal health of ducks, which was attributed to the improvement in cecal microbiota, intestinal morphology, and barrier function.

A Broad-Spectrum Phage Endolysin (LysCP28) Able to Remove Biofilms and Inactivate Clostridium perfringens Strains.

Clostridium perfringens is a gram-positive, anaerobic, spore-forming bacterium capable of producing four major toxins which cause disease symptoms and pathogenesis in humans and animals. C. perfringens strains carrying enterotoxins can cause food poisoning in humans and are associated with meat consumption. An endolysin, named LysCP28, is encoded by orf28 from C. perfringens bacteriophage BG3P. This protein has an N-terminal glycosyl-hydrolase domain (lysozyme) and a C-terminal SH3 domain. Purified LysCP28 (38.8 kDa) exhibited a broad spectrum of lytic activity against C. perfringens strains (77 of 96 or 80.21%), including A, B, C, and D types, isolated from different sources. Moreover, LysCP28 (10 µg/mL) showed high antimicrobial activity and was able to lyse 2 × 107 CFU/mL C. perfringens ATCC 13124 and C. perfringens J21 (animal origin) within 2 h. Necessary due to this pathogenic bacterium's ability to form biofilms, LysCP28 (18.7 µg/mL) was successfully evaluated as an antibiofilm agent in both biofilm removal and formation inhibition. Finally, to confirm the efficacy of LysCP28 in a food matrix, duck meat was contaminated with C. perfringens and treated with endolysin (100 µg/mL and 50 µg/mL), which reduced viable bacteria by 3.2 and 3.08 units-log, respectively, in 48 h at 4 °C. Overall, the endolysin LysCP28 could potentially be used as a biopreservative to reduce C. perfringens contamination during food processing.

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 of Gut Microbiota and Intestinal Barrier Dysfunction in Pigs with Pulmonary Inflammation Induced by Mycoplasma hyorhinis Infection.

Lung inflammation induced by Mycoplasma hyorhinis infection accounts for significant economic losses in the swine industry. Increasing evidence suggests that there is cross talk between the lungs and the gut, but little is known about the effect of the lung inflammation caused by M. hyorhinis infection on gut microbiota and intestinal barrier function. Here, we investigated changes in the fecal microbiotas of pigs with M. hyorhinis infection and the microbial regulatory role of such infection in intestinal barrier function. We infected pigs with M. hyorhinis and performed 16S rRNA gene sequencing analyses of fecal samples, data-independent acquisition (DIA) quantitative proteomic analyses of intestinal mucosa, and analyses of barrier dysfunction indicators in serum. We found that pigs with M. hyorhinis infection exhibit lung and systemic inflammation, as reflected by the histopathological changes and activation of the TLR4/MyD88/NF-κB p65 signaling pathway in lung tissue, as well as the increased concentrations of serum inflammatory cytokines. Gut microbiotas tended to become disturbed, as evidenced by the enrichment of opportunistic pathogens. The increased diamine oxidase activities and d-lactate concentrations in serum and the decreased relative mRNA expression of Occludin, ZO-1, and Mucin2 indicated the impairment of intestinal barrier function. Quantitative proteomic analyses showed a variety of altered proteins involved in immunomodulatory and inflammatory functions. There was a positive correlation between the abundance of opportunistic pathogens and inflammatory-cytokine concentrations, as well as intestinal immunomodulatory proteins. Our results suggest that lung inflammation induced by M. hyorhinis infection can contribute to the dysbiosis of gut microbiota and intestinal barrier dysfunction, and dysbiosis of gut microbiota was associated with systemic inflammation and intestinal immune status. IMPORTANCE Cumulative evidence suggests that bacterial pneumonia may contribute to the dysbiosis of the gut microbiota and other gastrointestinal symptoms. Our experiment has demonstrated that lung inflammation induced by M. hyorhinis infection was associated with gut microbiota dysbiosis and intestinal barrier dysfunction, which may provide a theoretical basis for exploring the gut-lung axis based on M. hyorhinis infection.

Genome Sequence of Salmonella enterica Serovar Typhimurium Phage SAP12.

Here, we report the genome of phage SAP012, which was isolated against Salmonella enterica serovar Typhimurium. The SAP012 genome is 59,618 bp, with a G+C content of 56.2% and with no antibiotic resistance or virulence genes, and is quite similar at the nucleotide level to a number of previously sequenced Salmonella phage genomes, e.g., GenBank accession numbers KM366098.1 and KC139515.1.

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.

Non-coding RNA regulates phage sensitivity in Listeria monocytogenes.

Non-coding RNAs (ncRNAs) have gained increasing attention as their diverse roles in virulence and environmental stress in Listeria monocytogenes have become clearer. The ncRNA rliB is an atypical member of the CRISPR family, conserved at the same genomic locus in all analyzed L. monocytogenes genomes and also in other Listeria species. In this study, rliB defective mutants (Lm3-22-ΔrliB) were constructed by homologous recombination. The growth cycle of Lm3-22-ΔrliB mutants was slower than that of wild-type Lm3-22. The sensitivity of Lm3-22-ΔrliB to the Listeria phage vB-LmoM-SH3-3 was significantly increased, and the efficiency of plaque formation was enhanced by 128 fold. Compared with wild type, the adhesion and invasion of Lm3-22-ΔrliB decreased significantly (9.3% and 1.33%, respectively). After 4 hours of infection, the proliferation of Lm3-22-ΔrliB in RAW264.7 cells also decreased significantly. Transcription level of invasion-related surface proteins showed that the internalin genes lmo0610 and lm0514, and the peptidoglycan binding protein gene lmo1799 in Lm3-22-ΔrliB were significantly increased. In addition, after interaction with phage, the transcription levels of inlA, lmo0610, lmo1799, lmo2085, and lmo0514 in Lm3-22-ΔrliB cells were significantly upregulated, while inlB was downregulated, compared with Lm3-22 control group with phage treatment. Therefore, rliB deletion effectively regulated the interaction between Listeria and phage, weaken its invasion ability, and provided a new theoretical basis for biocontrol of phage.

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.

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.

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.

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.

Morphologic and genomic characterization of a broad host range Salmonella enterica serovar Pullorum lytic phage vB_SPuM_SP116.

For effective use of phages as antimicrobial agents for controlling multidrug resistant S. Pullorum, it is important to understand phage biology. A lytic S. Pullorum phage was isolated and characterized from chicken feces, and its whole genome was sequenced and analyzed. A new lytic phage-vB_SPuM_SP116 (in brief SP116)- isolated and characterized using S. Pullorum SPu-116 as its host belongs to Myoviridae A1 group. Phage SP116 had a lytic effect on 27 of 37 (72.9%) different serotypes of clinical Salmonella strains. It showed a high bactericidal activity in killing all pathogens in cultures containing 5 × 105 cfu/mL and achieved more than 6.58 and 5.97 log unit reductions in cultures containing 5 × 106 cfu/mL and 5 × 107 cfu/mL, respectively. The one-step growth curve showed that the burst size was up to 118 pfu/bacterial cell. Complete genome sequence analysis revealed a linear, double-stranded DNA genome of 87,510 bp with an average G + C content of 38.84%, including 128 predicted open reading frames (ORFs) and 22 tRNA genes. SP116 was classified as a Felix O1 virus based upon the general phage characterization and the genomic information. Regarding its high efficacy in preventing especially S. Pullorum infection and its lack of any bacterial virulence, antimicrobial resistance, and lysogenesis genes, it could be a potential alternative candidate for the treatment of S. Pullorum infections.

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.

Testosterone disruptor effect and gut microbiome perturbation in mice: Early life exposure to doxycycline.

Veterinary tetracyclines drugs are emerging organic pollutants detected at high concentrations in the urine of school children and a potential public health risk. However, the implications of early-life exposure to tetracyclines on testosterone production, being new endocrine disruptors, remain unknown. We investigated whether the early-life exposure to low-doxycycline, a widely used tetracycline, on mitochondria dysfunction and testosterone disruption in Leydig cells in vitro and in vivo. Next, we determined the mRNA levels of testis cells markers for early-life exposure to low-doxycycline outcomes of testis health in later-life. Finally, we compared the weight gain performance exposed to low- and therapeutic-doses through 15 weeks and examined the role of the microbiota during development. Our results showed doxycycline disturbed steroidogenesis process by mitochondrial dysfunction in mouse Leydig tumor cell line (MLTC-1) cells in vitro. Leydig cells mitochondrial function was disrupted by early-life exposure to low-doxycycline from birth to 49 days, causing testosterone deficiency and decreased quality of the sperm in mice. Early-life exposure to low-doxycycline significantly altered the mRNA levels of key genes in Leydig cells (Cyp11a1, Cyp17a1 and 17ß-HSD) and spermatogenic cells (Grfal, Plzf, and Stra8) in later-life in mice. Subchronic low- and therapeutic-doses doxycycline changed gut microbiota differences in diversity reduction and compositional alteration. Moreover, the weight gain effects of doxycycline were only observed in low-dose in male mice. Overall, these results provide insight into the effects of doxycycline on both testis and gut microbiota health. The results provide insight that environmental antibiotics are needed additional research to classify as ECDs.

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.

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.