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Multidrug-resistant (MDR) Salmonella serovars are considered a significant threat to veterinary and public health. Developing new antimicrobial compounds that can treat the infection caused by these notorious pathogens is a big challenge. Bacteriophages can be adsorbed on and inhibit the growth of bacteria, providing optimal and promising alternatives to chemical antimicrobial compounds against foodborne pathogens due to their abundance in nature and high host specificity. The objective of the current study was to isolate and characterize new phages from poultry farms and sewage and to evaluate their efficacy against S. Enteritidis isolates. The study reports three lytic phages designated as ϕSET1, ϕSET2, and ϕSET3 isolated from poultry carcasses and sewage samples in Qalubiya governorate Egypt. The effectiveness of phages was evaluated against multidrug-resistant S. Enteritidis strains. Electron microscopy showed that these phages belong to the Siphoviridae family. Phages were tested against 13 bacterial strains to determine their host range. They could infect four S. Enteritidis and one S. Typhimurium; however, they did not infect other tested bacterial species, indicating their narrow infectivity. The bacteriophage's single-step growth curves revealed a latent period of 20 min for ϕSET1 and 30 min for ϕSET2 and ϕSET3. The isolated Salmonella phages prevented the growth of S. Enteritidis for up to 18 hrs. The findings revealed that Salmonella phages could be used as alternative natural antibacterial compounds to combat infection with MDR S. Enteritidis in the poultry industry and represent a step forward to using large panels of phages for eliminating Salmonella from the food chain.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms. However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces, the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong ErmE promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health.

Abstract Chassis strains, derived from Streptomyces coelicolor M145, deleted for one or more of its four main specialized metabolites biosynthetic pathways (CPK, CDA, RED and ACT), in various combinations, were constructed for the heterologous expression of specialized metabolites biosynthetic pathways of various types and origins. To determine consequences of these deletions on the metabolism of the deleted strains comparative lipidomic and metabolomic analyses of these strains and of the original strain were carried out. These studies unexpectedly revealed that the deletion of the peptidic clusters, RED and/or CDA, in a strain deleted for the ACT cluster, resulted into a great increase in the triacylglycerol (TAG) content, whereas the deletion of polyketide clusters, ACT and CPK had no impact on TAG content. Low or high TAG content of the deleted strains was correlated with abundance or paucity in amino acids, respectively, reflecting high or low activity of oxidative metabolism. Hypotheses based on what is known on the bio‐activity and the nature of the precursors of these specialized metabolites are proposed to explain the unexpected consequences of the deletion of these pathways on the metabolism of the bacteria and on the efficiency of the deleted strains as chassis strains.

Abstract Background Hyaluronic acid (HA) has gained significant attention due to its unique physical, chemical, and biological properties, making it widely used in various industries. This study aimed to screen bacterial isolates for HA production, characterize favorable fermentation conditions, and evaluate the inhibitory effect of bacterial HA on cancer cell lines. Results A total of 108 bacterial isolates from diverse sources were screened for HA production using HPLC, turbidimetric, and carbazole determination methods. Among the HA-producing isolates, Klebsiella pneumoniae H15 isolated from an animal feces sample, was superior in HA production. The strain was characterized based on its morphological, cultural, and biochemical characteristics. Molecular identification using 16S rDNA sequencing and phylogenetic analysis confirmed its identity. Fermentation conditions, including pH, temperature, time, and agitation rate, were optimized to maximize HA production. The basal medium, comprising sucrose (7.0%) as carbon source and combined yeast extract with peptone (1.25% each) as nitrogen substrate, favored the highest HA production at pH 8.0, for 30 h, at 30 °C, under shaking at 180 rpm. The average maximized HA concentration reached 1.5 g L−1. Furthermore, bacterial HA exhibited a significant inhibitory effect on three cancer cell lines (MCF-7, HepG-2 and HCT), with the lowest concentration ranging from 0.98–3.91 µg mL−1. Conclusions K. pneumoniae H15, isolated from animal feces demonstrated promising potential for HA production. The most favorable fermentation conditions led to a high HA production. The inhibitory effect of bacterial HA on cancer cell lines highlights its potential therapeutic applications. These findings contribute to a broader understanding and utilization of HA in various industries and therapeutic applications.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms. However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces, the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong ErmE promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health.

The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. The aim of the current study was to determine the prevalence of Aeromonas spp. in raw fish markets and humans in Zagazig, Egypt; identify the factors that contribute to virulence; determine the isolates’ profile of antibiotic resistance; and to elucidate the ability of Aeromonas spp. to form biofilms. The examined samples included fish tissues and organs from tilapia (Oreochromis niloticus, n = 160) and mugil (Mugil cephalus, n = 105), and human skin swabs (n = 51) and fecal samples (n = 27). Based on biochemical and PCR assays, 11 isolates (3.2%) were confirmed as Aeromonas spp. and four isolates (1.2%) were confirmed as A. hydrophila. The virulence genes including haemolysin (hyl A) and aerolysin (aer) were detected using PCR in A. hydrophila in percentages of 25% and 50%, respectively. The antimicrobial resistance of Aeromonas spp. was assessed against 14 antibiotics comprising six classes. The resistance to cefixime (81.8%) and tobramycin (45.4%) was observed. The multiple antibiotic resistance (MAR) index ranged between 0.142–0.642 with 64.2% of the isolates having MAR values equal to 0.642. Biofilm formation capacity was assessed using a microtiter plate assay, and two isolates (18.1%) were classified as biofilm producers. This study establishes a baseline for monitoring and controlling the multidrug-resistant Aeromonas spp. and especially A. hydrophila in marine foods consumed in our country to protect humans and animals.

The challenge of antibiotic resistance has gained much attention in recent years due to the rapid emergence of resistant bacteria infecting humans and risking industries. Thus, alternatives to antibiotics are being actively searched for. In this regard, bacteriophages and their enzymes, such as endolysins, are a very attractive alternative. Endolysins are the lytic enzymes, which are produced during the late phase of the lytic bacteriophage replication cycle to target the bacterial cell walls for progeny release. Here, we cloned, expressed, and purified LysZC1 endolysin from Pseudomonas phage ZCPS1. The structural alignment, molecular dynamic simulation, and CD studies suggested LysZC1 to be majorly helical, which is highly similar to various phage-encoded lysozymes with glycoside hydrolase activity. Our endpoint turbidity reduction assay displayed the lytic activity against various Gram-positive and Gram-negative pathogens. Although in synergism with EDTA, LysZC1 demonstrated significant activity against Gram-negative pathogens, it demonstrated the highest activity against Bacillus cereus. Moreover, LysZC1 was able to reduce the numbers of logarithmic-phase B. cereus by more than 2 log10 CFU/mL in 1 h and also acted on the stationary-phase culture. Remarkably, LysZC1 presented exceptional thermal stability, pH tolerance, and storage conditions, as it maintained the antibacterial activity against its host after nearly one year of storage at 4 °C and after being heated at temperatures as high as 100 °C for 10 min. Our data suggest that LysZC1 is a potential candidate as a therapeutic agent against bacterial infection and an antibacterial bio-control tool in food preservation technology.

Chassis strains, derived from Streptomyces coelicolor M145, deleted for one or more of its four main specialized metabolites biosynthetic pathways (CPK, CDA, RED and ACT), in various combinations, were constructed for the heterologous expression of specialized metabolites biosynthetic pathways of various types and origins. To determine consequences of these deletions on the metabolism of the deleted strains comparative lipidomic and metabolomic analyses of these strains and of the original strain were carried out. These studies unexpectedly revealed that the deletion of the peptidic clusters, RED and/or CDA, in a strain deleted for the ACT cluster, resulted into a great increase in the triacylglycerol (TAG) content, whereas the deletion of polyketide clusters, ACT and CPK had no impact on TAG content. Low or high TAG content of the deleted strains was correlated with abundance or paucity in amino acids, respectively, reflecting high or low activity of oxidative metabolism. Hypotheses based on what is known on the bio‐activity and the nature of the precursors of these specialized metabolites are proposed to explain the unexpected consequences of the deletion of these pathways on the metabolism of the bacteria and on the efficiency of the deleted strains as chassis strains. [ABSTRACT FROM AUTHOR]

This study investigated the in vitro and in vivo anti-hepatocellular carcinoma activity of pseurotin A isolated from the n-butanol extract of Aspergillus fumigatus Fr. AUMC 8002 (nBE-AF).The in vitro anticancer activity of nBE-AF was measured against a human hepatocellular carcinoma cell line (HepG2) using a sulforhodamine-B (SRB) assay. The intraperitoneal median lethal dose (LD50) of nBE-AF was determined in rats. Hepatocellular carcinoma was induced in rats by a single intraperitoneal injection of diethylnitrosamine (DEN) (200 mg/kg b.wt.) followed by subcutaneous injections of carbon tetrachloride (CCl4) (3 ml/kgb.wt.) weekly for 6 weeks. After administration of these carcinogens, 1/10 and 1/20 LD50 doses of nBE-AF were administered intraperitoneally daily. NBE-AF exhibited significant cytotoxic activity against HepG2 cells. Administration of DEN and CCl4 significantly elevated the serum levels of liver function and tumour markers and significantly downregulated tumour necrosis factor-α gene expression. Moreover, DEN and CCl4 decreased immunohistochemical Bax expression and increased Bcl-2 expression in the liver. Co-treatment with nBE-AF mitigated the DEN+CCl4-induced alterations in a dose-dependent manner. Histopathological evaluation of the liver substantiated the above biochemical results. These results confirmed that nBE-AF, via its major isolated secondary metabolite, pseurotin A, exerted an anti-hepatocarcinogenic effect and could be used as a chemopreventive agent for hepatocellular carcinoma.awareness of the role of mycobacteria in infections associated with exposure to aquarium fish. Key words: Aspergillus fumigatus extract; pseurotin A; hepatocellular carcinoma; anticancer; cytotoxic PSEUROTIN A IZ GLIVE Aspergillus fumigatus Fr. AUMC 8002 ZAVIRA RAST KARCINOMA JETER IN VITRO IN IN VIVO Povzetek: V študiji so raziskali in vitro in in vivo protirakasto aktivnost pseurotina A, pridobljenega iz n-butanolnega izvlečka glive Aspergillus fumigatus Fr. AUMC 8002 (nBE-AF), na karcinom jeter. In vitro protirakasta aktivnost nBE-AF je bila spremljana pri humani celični liniji jetrnega karcinoma (HepG2) z uporabo analize sulforhodamin-B (SRB). Pri podganah so intraperitonealno določili povprečni smrtni odmerek (LD ) nBE-AF. Jetrni karcinom je bil pri podganah sprožen z enkratno intraperitonealno injekcijo dietilnitrozamina (DEN) (200 mg/kg telesne teže), čemur so sledile podkožne injekcije ogljikovega tetraklorida (CCl 50 ) (3 ml/ kg) enkrat tedensko 6 tednov. Po dodajanju teh rakotvornih snovi so 1/10 in 1/20 odmerka LD nBE-AF aplicirali vsak dan intraperitonealno. NBE-AF je pokazal pomembno citotoksično aktivnost proti celicam HepG2. Uporaba DEN in CCl4 je značilno povečala serumske vrednosti jetrnih funkcij in tumorskih markerjev ter znatno zmanjšala izražanje gena za faktor tumorske nekroze α (TNF- α). Poleg tega sta DEN in CCl 4 50 zmanjšala imunohistokemično izražanje gena Bax in povečala izražanje gena Bcl-2 v jetrih. Sočasno zdravljenje z nBE-AF je zmanjšalo z DEN + CCl sprožene spremembe v jetrih v povezavi z velikostjo odmerka. Histopatološka ocena jeter je potrdila zgoraj navedene biokemične rezultate. Rezultati so potrdili, da je nBE-AF preko svojega glavnega izoliranega sekundarnega metabolita pseurotina A izkazal protirakasti učinek na jetra in bi se lahko v prihodnosti morda uporabljal kot zdravilo za zdravljenje karcinoma jeter. Ključne besede: izvleček Aspergillus fumigatus; pseurotin A; karcinom jeter; protirakasto; citotoksično

Phage therapy is an alternative treatment to antibiotics that can overcome multi-drug resistant bacteria. In this study, we aimed to isolate and characterize lytic bacteriophages targeted against Enterococcus faecalis isolated from root canal infections obtained from clinics at the Faculty of Dentistry, Ismalia, Egypt. Bacteriophage, vB_ZEFP, was isolated from concentrated wastewater collected from hospital sewage. Morphological and genomic analysis revealed that the phage belongs to the Podoviridae family with a linear double-stranded DNA genome, consisting of 18,454, with a G + C content of 32.8%. Host range analysis revealed the phage could infect 10 of 13 E. faecalis isolates exhibiting a range of antibiotic resistances recovered from infected root canals with efficiency of plating values above 0.5. One-step growth curves of this phage showed that it has a burst size of 110 PFU per infected cell, with a latent period of 10 min. The lytic activity of this phage against E. faecalis biofilms showed that the phage was able to control the growth of E. faecalis in vitro. Phage vB_ZEFP could also prevent ex-vivo E. faecalis root canal infection. These results suggest that phage vB_ZEFP has potential for application in phage therapy and specifically in the prevention of infection after root canal treatment.

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Background: Hyaluronic acid (HA) has gained significant attention due to its unique physical, chemical, and biological properties, making it widely used in various industries. This study aimed to screen bacterial isolates for HA production, characterize favorable fermentation conditions, and evaluate the inhibitory effect of bacterial HA on cancer cell lines. Results: A total of 108 bacterial isolates from diverse sources were screened for HA production using HPLC, turbidimetric, and carbazole determination methods. Among the HA-producing isolates, Klebsiella pneumoniae H15 isolated from an animal feces sample, was superior in HA production. The strain was characterized based on its morphological, cultural, and biochemical characteristics. Molecular identification using 16S rDNA sequencing and phylogenetic analysis confirmed its identity. Fermentation conditions, including pH, temperature, time, and agitation rate, were optimized to maximize HA production. The basal medium, comprising sucrose (7.0%) as carbon source and combined yeast extract with peptone (1.25% each) as nitrogen substrate, favored the highest HA production at pH 8.0, for 30 h, at 30 °C, under shaking at 180 rpm. The average maximized HA concentration reached 1.5 g L−1. Furthermore, bacterial HA exhibited a significant inhibitory effect on three cancer cell lines (MCF-7, HepG-2 and HCT), with the lowest concentration ranging from 0.98–3.91 µg mL−1. Conclusions: K. pneumoniae H15, isolated from animal feces demonstrated promising potential for HA production. The most favorable fermentation conditions led to a high HA production. The inhibitory effect of bacterial HA on cancer cell lines highlights its potential therapeutic applications. These findings contribute to a broader understanding and utilization of HA in various industries and therapeutic applications. [ABSTRACT FROM AUTHOR]

A P2-like phage ϕRSY1 infecting the phytopathogen Ralstonia solanacearum was isolated and characterized. The 40-kb genome of ϕRSY1 showed high sequence similarity to the Ralstonia phage ϕRSA1 and the GMI1000 prophage ϕRSX. The major genomic differences between these phages were the different orientation of the int gene and the gene content close to the cosL. ϕRSY1 and ϕRSX use a 15-base 3′ portion of the serine tRNA(GGA) gene as attB, while ϕRSA1 uses a 45-base 3′ portion of the arginine tRNA(CCG) gene. The different orientation of int in the genomes means that the gene arrangements in the prophage states are reversed in ϕRSY1 and ϕRSA1. Several putative gene products of ϕRSY1 may affect the bacterium’s fitness. ϕRSY1 contains an open reading frame (ORF) that seems to encode a protein similar to Vgr in the type VI secretion system of various bacterial species. ϕRSY1 lysogens showed phenotypic changes including enhanced twitching motility, large colony formation, and easy aggregation of cells, suggesting involvement of this ORF in the changes. In view of these phage gene arrangements, we surveyed prophages in the genomes of various R. solanacearum strains and found that the P2-like phages of R. solanacearum (14 phages) consist of two major groups: the ϕRSY1-type and the ϕRSA1-type. The relationships and evolution of these P2-like phages inferred from our data are discussed in detail.