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Background and Aim: The dearth of new antibiotics necessitates alternative approaches for managing infections caused by resistant superbugs. This study aimed to evaluate the lytic potential of the purified bacteriophage PKp-V1 against extended-spectrum β-lactamase (ESBL) harboring hypervirulent Klebsiella pneumoniae (hvKp)-K1 recovered from veterinary specimens. Materials and Methods: A total of 50 samples were collected from various veterinary specimens to isolate K. pneumoniae, followed by antimicrobial susceptibility testing and molecular detection of various virulence and ESBL genes. Multilocus sequence typing of the isolates was performed to identify prevalent sequence types. The bacteriophages were isolated using the double-agar overlay method and characterized using transmission electron microscopy, spot tests, plaque assays, stability tests, and one-step growth curve assays. Results: Among 17 (34%) confirmed K. pneumoniae isolates, 6 (35%) were hvKp, whereas 13 (76%) isolates belonging to the K1 type were positive for the wzy (K1) virulence gene. All (100%) hvKp isolates exhibited the allelic profile of ST258. Overall, PKp-V1 exhibited an 88 % (15/17; (p ≤ 0.05) host range, among which all (100 %; p ≤ 0.01) hvKp isolates were susceptible to PKp-V1. PKp-V1 exhibited a lytic phage titer of 2.4 × 108 plaque forming unit (PFU)/mL at temperatures ranging from 25°C to 37°C. The lytic phage titers of PKp-V1 at pH = 8 and 0.5% chloroform were 2.1 × 108 PFU/mL and 7.2 × 109 PFU/mL, respectively. Conclusion: Although the incidence of ESBL-infected K. pneumoniae in veterinary settings is worrisome, PKp-V1 phages showed considerable lytic action against the host bacterium, indicating the potential of PKp-V1 as a possible alternative therapeutic option against MDR K. pneumoniae.

Contamination of agricultural and industrial products by Aspergillus section Circumdati becomes a true problem, especially, because a lot of species in this section can yield ochratoxins and other toxins. In this study, morphological criteria and partial calmodulin gene were used to identify 34 strains belonging to Aspergillus section Circumdati isolated from Vitis vinifera and Calotropis procera plants. The population is characterised by A. insulicola, A. ochraceopetaliformis, A. ochraceus, and A. pseudoelegans. The polyketide synthase (pks) gene involved in ochratoxins (OTA) production was investigated by Aopks1, 2 and AoLc35–12L, R primers. Fifteen strains belonging to A. ochraceus were positive for pks gene, whilst A. insulicola, A. ochraceopetaliformis, and A. pseudoelegans were negative. All tested strains were able to produce ochratoxin A with different levels of 0.020–53 ppm except one isolate of A. ochraceus (IAEMAo6). The green synthesised ZnO-NPs have a significant inhibitory effect on OTA production by A. insulicola and A. pseudoelegans.

This study aimed to identify Salmonella species in two systems for quail raising (deep litter system and cages system). Determination of antibiotic susceptibility, β-lactamase resistance genes profile and susceptibility to chitosan, thyme extract and S. cerevisiae was examined. Fifteen Salmonella isolates were isolated from eighty cloacal swabs examined, with a higher incidence from those of deep litter systems than cage housing systems. Regarding the environmental samples, Salmonella was higher in the deep litter system than in cage housing. Serological identification of 27 salmonella isolates typed them into 8 Salmonella spp. including S. Tsevie, S. Infantis, S. Kentucky, S. Typhimurium, S. Larochelle, S. Molade, S. Enteritidis and S. Labadi. The profile of β-lactamase-resistance genes revealed the presence of blaTEM and blaSHV genes in S. Typhimurium and S. Infantis; blaTEM and blaCTX-M1 genes in S. Enteritidis and S. Molade, while S. Kentucky and S. Tsevie were positive for the blaCTX-M1 gene only. S. Larochelle was negative for all of them. Thyme and chitosan extract showed that S. cerevisiae had the highest inhibitory activity against the tested strains. In conclusion, farms with superior biosecurity and hygiene practices will reduce the risk of pathogens and eliminate the need for prophylactic antibiotics, thereby preventing antibiotic resistance.

IntroductionAeromonas hydrophila and methicillin-resistant Staphylococcus aureus (MRSA) are potent bacterial pathogens posing major hazards to human health via consuming fish harboring these pathogens or by cross-contamination beyond the contaminated environment. The aim of this study was to determine risk variables associated with the presence of certain pathogenic bacteria from Mugil cephalus fish in retail markets in Egypt. The virulence genes of A. hydrophila and S. aureus were also studied. Furthermore, the antibiotic sensitivity and multidrug resistance of the microorganisms were evaluated.MethodsIn a cross-sectional investigation, 370 samples were collected from mullet skin and muscle samples, washing water, fish handlers, knives, and chopping boards. Furthermore, fish handlers’ public health implications were assessed via their response to a descriptive questionnaire.ResultsS. aureus and Aeromonas species dominated the investigated samples with percentages of 26.76% and 30.81%, respectively. Furthermore, A. hydrophila and MRSA were the predominant recovered bacterial pathogens among washing water and knives (53.85% and 46.66%, respectively). The virulence markers aerA and hlyA were found in 90.7% and 46.5% of A. hydrophila isolates, respectively. Moreover, the virulence genes nuc and mec were prevalent in 80% and 60% of S. aureus isolates, respectively. Antimicrobial susceptibility results revealed that all A. hydrophila isolates were resistant to amoxicillin and all MRSA isolates were resistant to amoxicillin and ampicillin. Remarkably, multiple drug resistance (MDR) patterns were detected in high proportions in A. hydrophila (88.37%) and MRSA (100%) isolates. The prevalence of Aeromonas spp. and S. aureus had a positive significant correlation with the frequency of handwashing and use of sanitizer in cleaning of instruments. MRSA showed the highest significant prevalence rate in the oldest age category.ConclusionThe pathogenic bacteria recovered in this study were virulent and had a significant correlation with risk factors associated with improper fish handling. Furthermore, a high frequency of MDR was detected in these pathogenic bacteria, posing a significant risk to food safety and public health.

IntroductionThere is an urgent need to develop therapeutic options for biofilm-producing Staphylococcus aureus (S. aureus). Therefore, the renewed interest in essential oils (EOs), especially carvacrol, linalool and eugenol, has attracted the attention of our research group.MethodsMultidrug resistance and multivirulence profiles in addition to biofilm production of S. aureus strains isolated from cows with mastitis were evaluated using both phenotypic and genotypic methods. The antimicrobial and antibiofilm activities of EOs were tested using both in vitro and molecular docking studies. Moreover, the interactions between commonly used antibiotics and the tested EOs were detected using the checkerboard method.ResultsWe found that all our isolates (n= 37) were biofilm methicillin resistant S. aureus (MRSA) producers and 40.5% were vancomycin resistant S. aureus (VRSA). Unfortunately, 73 and 43.2% of the recovered MRSA isolates showed multidrug resistant (MDR) and multivirulence patterns, respectively. The antimicrobial activities of the tested EOs matched with the phenotypic evaluation of the antibiofilm activities and molecular docking studies. Linalool showed the highest antimicrobial and antibiofilm activities, followed by carvacrol and eugenol EOs. Fortunately, synergistic interactions between the investigated EOs and methicillin or vancomycin were detected with fractional inhibitory concentration index (FICI) values ≤ 0.5. Moreover, the antimicrobial resistance patterns of 13 isolates changed to sensitive phenotypes after treatment with any of the investigated EOs. Treatment failure of bovine mastitis with resistant S. aureus can be avoided by combining the investigated EOs with available antimicrobial drugs.ConclusionWe hope that our findings can be translated into a formulation of new pharmaceutical dosage forms against biofilm-producing S. aureus pathogens.

ABSTRACT Control and management of life-threatening bacterial and fungal infections are a global health challenge. Despite advances in antimicrobial therapies, treatment failures for resistant bacterial and fungal infections continue to increase. We aimed to repurpose the anthelmintic drug rafoxanide for use with existing therapeutic drugs to increase the possibility of better managing infection and decrease treatment failures. For this purpose, we evaluated the antibacterial and antifungal potential of rafoxanide. Notably, 70% (70/100) of bacterial isolates showed multidrug resistance (MDR) patterns, with higher prevalence among human isolates (73.5% [50/68]) than animal ones (62.5% [20/32]). Moreover, 22 fungal isolates (88%) were MDR and were more prevalent among animal (88.9%) than human (87.5%) sources. We observed alarming MDR patterns among bacterial isolates, i.e., Klebsiella pneumoniae (75% [30/40; 8 animal and 22 human]) and Escherichia coli (66% [40/60; 12 animal and 28 human]), and fungal isolates, i.e., Candida albicans (86.7% [13/15; 4 animal and 9 human]) and Aspergillus fumigatus (90% [9/10; 4 animal and 5 human]), that were resistant to at least one agent in three or more different antimicrobial classes. Rafoxanide had antibacterial and antifungal activities, with minimal inhibitory concentration (MICs) ranging from 2 to 128 μg/mL. Rafoxanide at sub-MICs downregulated the mRNA expression of resistance genes, including E. coli and K. pneumoniae blaCTX-M-1, blaTEM-1, blaSHV, MOX, and DHA, C. albicans ERG11, and A. fumigatus cyp51A. We noted the improvement in the activity of β-lactam and antifungal drugs upon combination with rafoxanide. This was apparent in the reduction in the MICs of cefotaxime and fluconazole when these drugs were combined with sub-MIC levels of rafoxanide. There was obvious synergism between rafoxanide and cefotaxime against all E. coli and K. pneumoniae isolates (fractional inhibitory concentration index [FICI] values ≤ 0.5). Accordingly, there was a shift in the patterns of resistance of 16.7% of E. coli and 22.5% of K. pneumoniae isolates to cefotaxime and those of 63.2% of C. albicans and A. fumigatus isolates to fluconazole when the isolates were treated with sub-MICs of rafoxanide. These results were confirmed by in silico and mouse protection assays. Based on the in silico study, one possible explanation for how rafoxanide reduced bacterial resistance is through its inhibitory effects on bacterial and fungal histidine kinase enzymes. In short, rafoxanide exhibited promising results in overcoming bacterial and fungal drug resistance. IMPORTANCE The drug repurposing strategy is an alternative approach to reducing drug development timelines with low cost, especially during outbreaks of disease caused by drug-resistant pathogens. Rafoxanide can disrupt the abilities of bacterial and fungal cells to adapt to stress conditions. The coadministration of antibiotics with rafoxanide can prevent the failure of treatment of both resistant bacteria and fungi, as the resistant pathogens could be made sensitive upon treatment with rafoxanide. From our findings, we anticipate that pharmaceutical companies will be able to utilize new combinations against resistant pathogens.

Nanocomposites based on hydroxyapatite (HAP) are fabricated with/without combining graphene oxide (GO) and silver oxide (Ag2O). All compositions are structurally investigated using x-ray diffraction (XRD). In addition, the lattice parameters were estimated. Lattice constants a and c of hexagonal HAP symmetry are around 9.42 and 6.88 Å, respectively. Further, the lattice constant of Ag2O is around 4.736 Å. Moreover, Fourier-transform infrared (FTIR) spectroscopy is used besides X-ray photoelectron (XPS) to investigate the composition. The morphological study was done with the scanning electron microscope (SEM) and transmission electron microscope (TEM). In this regard, Ag2O is exhibited as nano-spherical grains, while HAP was shown in nanorod grains. The dimensions of HAP decreased from 39 to 16 to 34 and 7 nm for length and diameter. The average roughness parameter (Ra) increased from 23.3 to 67.5 nm from HAP to TNC. The parameters of average maximum height (Rtm), maximum peak height (Rp), and maximum valley depth (Rv) reached 439.3, 261.3, and 251.8 nm respectively. Further, the Ca/P ratio is detected with a value of 1.5. Besides, cell viability of osteoblast cell lines increased from 95.9 ± 3 to 101.2 ± 5%. In addition, the antibacterial behavior increased to 21.6 ± 2.3 and 21.1 ± 1.4 mm for TNC against Escherichia coli and Staphylococcus aureus, respectively.

Bone tissue engineering is the most used technology in recent years for repairing damaged bones. A novel heterogeneous biomaterial is prepared and studied to be suggested as a promising bone substitute biomaterial, based on three different nanoparticles: hydroxyapatite (HAP), boron oxide (B2O3), and graphene oxide (GO) a triple nanocomposite is designed HAP/B2O3/GO. This hybrid scaffold is analyzed with XRD to examine the crystalline structure of HAP/B2O3/GO and shows its crystal planes. Moreover, the interaction between the three nanoparticles and their distinctive active functional groups is illustrated with FTIR analysis. Further, TEM micrographs demonstrate the structural alteration and the uniform incorporation of HAP and B2O3 into GO sheets with an average length of 30 nm and diameter of 6 nm for HAP, and a size of 8 mm for B2O3. Furthermore, SEM micrographs reveal the porous structure of HAP/B2O3/GO with a grain size of 5–10 nm. The maximum roughness average value is obtained by HAP/B2O3/GO which is 11.04 nm. Besides, HAP/B2O3/GO nanocomposite shows the highest cell viability value of 96.1 ± 1.2%. The bacterial toxicity of HAP/B2O3/GO is confirmed with the obtained inhibition zone against Escherichia coli and Staphylococcus aureus of 15.7 ± 1.1 mm and 16.2 ± 0.8 mm respectively. In addition, the hardness of the triple composite is improved showing a maximum value of 3.7 ± 0.3 GPa. Distinctly, the addition of B2O3 to HAP and GO nanosheets alter the morphological properties and enhances the roughness parameters of HAP/B2O3/GO. Besides, HAP/B2O3/GO shows improved porosity, cell viability, and biodegradability. Hence, studying HAP/B2O3/GO nanocomposite with different scans and analyses reveals its porous integrated structure and shows an excellent antibacterial property, so that HAP/B2O3/GO composite can be suggested as a promising bone substitute biomaterial.

Objectives: Endophytic fungi are widely known to reside in plant tissues causing no harm to host plants or even no noticeable change. They may colonize host plants for a part of their life cycle or may complete the entire life cycle since host provide a variety of novel metabolites. Despite being in a close relationship with medicinal host plants, the diversity and metabolic spectrum of endophytic fungi have not been researched well. Their diversity and ecological niche as plant symbionts make them attractive targets in the search for novel biochemicals. Methods: We aimed to isolate, purify, and characterize the endophytic fungal population of medicinal host plants cultivated in Riyadh, Saudi Arabia. The goal was achieved through morphological identification and internal transcribed spacer (ITS) regions in rRNA analyses to determine phylogeny and diversity. Three aromatic and medicinal plants Jasminum sambac, Camellia sinensis, and Ocimum basilicum collected from the Riyadh region, Saudi Arabia were investigated. In total, 84 isolates were purified and they were grouped into 20 operational taxonomic units (OTUs) as per their sequences of ITS regions in rRNA. Results: Twenty species of endophytic fungi were grouped in 12 genera i.e. Neopestalotiopsis, Trichoderma, Fusarium, Colletotrichum, Myrothecium, Chaetomium, Alternaria, Phoma, Curvularia, Cladosporium, Neodidymelliopsis, and Aspergillus and all isolates belonged to Ascomycota phylum. J. sambac was found dominant among other and had a relative frequency of 27%. C. sinensis was next with 18.7% relative frequency. The diversity was prominently recorded in leaf organs over stem and roots while roots exhibited the lowest diversity. Isolates also produced indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase enzyme, and siderophores with variable magnitudes that could be assigned to their wide range of metabolic variations from species to species. Conclusion: Conclusively, J. sambac, C. sinensis, and O. basilicum are a good source of endophytic fungi with certain plant growth-promoting traits. Overall, Alternaria was found as the most predominant genus in terms of colonization rate. Further deternminations are required to screen the beneficial compounds released by these endophytic fungi.

Our study proposed a promising anti-fungal therapy to control Saprolegnia spp. infection in Nile tilapia (Oreochromis niloticus). A Moringa oleifera synthesized silver nanoparticles (MS-AgNPs) showed an in vitro anti-fungal activity against Saprolegnia spp. with an inhibition zone of 18 mm. A total of 220 O. niloticus (45 ± 0.56 g) were used in this study. A preliminary investigation was conducted by using 100 fish for determining the safe dose of MS-AgNPs which was found to be 0.6 mg/L. Another 120 fish were divided into four groups; the first (control) and second groups (G1 and G2) were non-infected and exposed to 0 and 0.6 mg/L MS-AgNPs, respectively. The third and fourth groups (G3 and G4) were infected with Saprolegnia spp. (1.5 × 104 spores/L) and exposed to 0 and 0.6 mg/L MS-AgNPs, respectively. The experiment lasted for 7 days. The infection with Saprolegnia spp. induced immunosuppression expressed by a marked decrease in immunoglobulin M, nitric oxide, complement 3, and lysozyme activity, with a 41 % survival rate. Additionally, a marked decline in the values of hepatic and branchial antioxidant enzymes (superoxide dismutase and catalase) in addition to serum total antioxidant capacity and protein profile indices. In contrast, Saprolegnia spp. infection elevated hepatic and branchial malondialdehyde, hepatic function indicators, and stress biomarkers (cholesterol and triglyceride) with up-regulation of the pro-inflammatory cytokines (IL-1ß and TNF-α), and the anti-inflammatory cytokines (IL-10 and TGF-β). Histopathological findings of the gills and musculature of infected fish showed severe hyperplasia of the epithelial lining with a fusion of secondary lamellae and necrotic myocytes admixed with edema. Interestingly, treatment of the challenged fish with MS-AgNPs at a dose of 0.6 mg/L could improve these variables by enhancing the histological architecture. According to these findings, MS-AgNPs have an ameliorative effect against the harmful effects of Saprolegnia spp. infection on immune-antioxidant status and hepatic functions indices. Consequently, MS-AgNPs at a dose of 0.6 mg/L is a promising option for use in the control of Saprolegnia spp. infection in aquaculture.

Plant-derived bioactive compounds with promising nutritional and therapeutic attributes (phytogenics) are among the top priorities in the aquaculture sector. Therefore, the impact of thymol (Thy) and/or thymoquinone (ThQ) on the growth, immune response antioxidant capacity, and Aeromonas sobria (A. sobria) resistance of Nile tilapia was investigated. Four fish groups were fed a control diet and three basal diets supplemented with 200 mg/kg diet of Thy or ThQ and a blend of both Thy and ThQ at a level of 200 mg/kg diet each. At the end of the feeding trial (12 weeks), the tilapias were challenged intraperitoneally with virulent A. sobria (2.5 × 108 CFU/mL) harboring aerolysin (aero) and hemolysin (hly) genes. The results revealed that tilapias fed diets fortified with a combination of Thy and ThQ displayed significantly enhanced growth rate and feed conversion ratio. Notably, the expression of the genes encoding digestive enzymes (pepsinogen, chymotrypsinogen, α-amylase and lipase) and muscle and intestinal antioxidant enzymes (glutathione peroxidase, catalase and superoxide dismutase) was significantly upregulated in Thy/ThQ-fed fish. An excessive inflammatory response was subsided more prominently in the group administrated Thy/ThQ as supported by the downregulation of il-β, il-6 and il-8 genes and in contrast, the upregulation of the anti-inflammatory il-10 gene. Remarkably, dietary inclusion of Thy/ThQ augmented the expression of autophagy-related genes, whilst it downregulated that of mtor gene improving the autophagy process. Furthermore, Thy/ThQ protective effect against A. sobria was evidenced via downregulating the expression of its aero and hly virulence genes with higher fish survival rates. Overall, the current study encouraged the inclusion of Thy/ThQ in fish diets to boost their growth rates, promote digestive and antioxidant genes expression, improve their immune responses and provide defense against A. sorbia infections with great economic benefits.

Escherichia coli (E.coli) found in retail chicken meat could be causing a wide range of infections in humans and constitute a potential risk. This study aimed to evaluate 60 E. coli isolates from retail chicken meat (n = 34) and human urinary tract infections (UTIs, n = 26) for phylogenetic diversity, presence of pathogenicity island (PAI) markers, antimicrobial susceptibility phenotypes, and antimicrobial resistance genes, and to evaluate their biofilm formation capacity. In that context, confirmed E.coli isolates were subjected to phylogrouping analysis using triplex PCR, antimicrobial susceptibility testing using the Kirby–Bauer disc diffusion method; PAI distribution was investigated by using two multiplex PCRs. Most of the chicken isolates (22/34, 64.7%) were identified as commensal E. coli (A and B1), while 12 isolates (35.3%) were classified as pathogenic virulent E. coli (B2 and D). Similarly, the commensal group dominated in human isolates. Overall, 23 PAIs were detected in the chicken isolates; among them, 39.1% (9/23) were assigned to group B1, 34.8% (8/23) to group A, 4.34% (1/23) to group B2, and 21.7% (5/23) to group D. However, 25 PAIs were identified from the human isolates. PAI IV536 was the most prevalent (55.9%, 69.2%) PAI detected in both sources. In total, 37 (61.7%) isolates of the chicken and human isolates were biofilm producers. Noticeably, 100% of E. coli isolates were resistant to penicillin and rifamycin. Markedly, all E. coli isolates displayed multiple antibiotic resistance (MAR) phenotypes, and the multiple antibiotic resistance index (MARI) among E. coli isolates ranged between 0.5 and 1. Several antibiotic resistance genes (ARGs) were identified by a PCR assay; the sul2 gene was the most prevalent (38/60, 63.3%) from both sources. Interestingly, a significant positive association (r = 0.31) between biofilm production and resistance to quinolones by the qnr gene was found by the correlation analysis. These findings were suggestive of the transmission of PAI markers and antibiotic resistance genes from poultry to humans or humans to humans through the food chain. To avoid the spread of virulent and multidrug-resistant E. coli, intensive surveillance of retail chicken meat markets is required.

The persistence of multidrug resistance among microorganisms has directed a mandate towards a hunt for the development of alternative therapeutic modalities. In this context, antimicrobial photodynamic therapy (aPDT) is sprouted as a novel strategy to mitigate biofilms and planktonic cells of pathogens. Nanoparticles (NPs) are reported with unique intrinsic and antimicrobial properties. Therefore, silver NPs (AgNPs) were investigated in this study to determine their ability to potentiate the aPDT of photosensitizer against Staphylococcus aureus and Pseudomonas aeruginosa. Biologically synthesized AgNPs were surface coated with methylene blue (MB) and studied for their aPDT against planktonic cells and biofilms of bacteria. The nano-conjugates (MB-AgNPs) were characterized for their size, shape and coated materials. MB-AgNPs showed significant phototoxicity against both forms of test bacteria and no toxicity was observed in the dark. Moreover, activity of MB-AgNPs was comparatively higher than that of the free MB, which concludes that MB-AgNPs could be an excellent alternative to combat antibiotic resistant bacteria.