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Bilal Aslam,1 Muhammad Hussnain Siddique,2 Abu Baker Siddique,1 Muhammad Shafique,1 Saima Muzammil,1 Mohsin Khurshid,1 Muhammad Hidayat Rasool,1 Moeed Ahmad,1 Tamoor Hamid Chaudhry,3 Afreenish Amir,3 Muhammad Salman,3 Zulqarnain Baloch,4 Norah A Alturki,5 Ahmad Alzamami6 1Department of Microbiology, Government College University Faisalabad, Faisalabad, Pakistan; 2Department of Bioinformatics & Biotechnology, Government College University Faisalabad, Faisalabad, Pakistan; 3Public Health Laboratories Division, National Institute of Health, Islamabad, Pakistan; 4Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, People’s Republic of China; 5Clinical Laboratory Science Department, College of Applied Medical Science, King Saud University, Riyadh, Saudi Arabia; 6Clinical Laboratory Science Department, College of Applied Medical Science, Shaqra University, AlQuwayiyah, Saudi ArabiaCorrespondence: Bilal Aslam, Department of Microbiology, Government College University Faisalabad, Faisalabad, Pakistan, Email drbilalaslam@gcuf.edu.pk Ahmad Alzamami, Clinical Laboratory Science Department, College of Applied Medical Science, Shaqra University, AlQuwayiyah, Saudi Arabia, Email aalzamami@su.edu.saBackground: The World Health Organization (WHO) has declared the multi-drug resistant (MDR) Klebsiella pneumoniae as one of the critical bacterial pathogens. The dearth of new antibiotics and inadequate therapeutic options necessitate finding alternative options. Bacteriophages are known as enemies of bacteria and are well-recognized to fight MDR pathogens.Methods: A total of 150 samples were collected from different clinical specimens through a convenient sampling technique. Isolation, identification, and antibiotic susceptibility testing (AST) of K. pneumoniae were done by standard and validated microbiological procedures. Molecular identification of virulence factors and antibiotic resistance genes (ARGs) was carried out through polymerase chain reaction (PCR) by using specific primers. For bacteriophage isolation, hospital sewage samples were processed for phage enrichment, purification, and further characterization ie, transmission electron microscopy (TEM) and stability testing, etc. followed by evaluation of the lytic potential of the phage.Results: Overall, a total of 41% of isolates of K. pneumoniae were observed as hypervirulent K. pneumoniae (hvKp). Among hvKp, a total of 12 (42%) were detected as MDR hvKp. A total of 37% of all MDR isolates were found resistant to colistin, and 66% of the colistin resistance isolates were recorded as mcr-1 positive. Isolated phage KpnM had shown lytic activity against 53 (79%) K. pneumoniae isolates. Remarkably, all 8 mcr-1 harboring MDR hvKp and non-hvKp isolates were susceptible to KpnM phage.Conclusion: Significant distribution of mcr-1 harboring hypervirulent Klebsiella pneumoniae was observed in clinical specimens, which is worrisome for the health system of the country. Characterized phage KpnM exhibited encouraging results and showed the lytic activity against the mcr-1 harboring hvKp isolates, which may be used as a prospective alternative control strategy to fight this ominous bacterium.Keywords: Klebsiella pneumoniae, bacteriophages, colistin, MDR, virulence

Tamoor Hamid Chaudhry,1,2 Bilal Aslam,1,2 Muhammad Imran Arshad,3 Roman Farooq Alvi,2 Saima Muzammil,2 Nafeesa Yasmeen,4 Muhammad Aamir Aslam,3 Mohsin Khurshid,2 Muhammad Hidayat Rasool,2 Zulqarnain Baloch1 1Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, People’s Republic of China; 2Department of Microbiology, Government College University Faisalabad, Faisalabad, Pakistan; 3Institute of Microbiology, University of Agriculture Faisalabad, Faisalabad, Pakistan; 4College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, People’s Republic of ChinaCorrespondence: Bilal Aslam; Zulqarnain Baloch Email drbilalaslam@gcuf.edu.pk; znbalooch@yahoo.comIntroduction: Intense livestock farming practices enforcing the farmers to use antibiotics as food supplements on a routine basis. Aberrant use of antibiotics is associated with the emergence of antibiotics resistance and resistant superbugs. Keeping in view the current scenario, the present study was designed for the first time from Pakistan with a specific aim to estimate the prevalence of the carbapenem-resistant Klebsiella pneumoniae in veterinary settings and the waste in Pakistan.Methods: A total of 138 samples from various veterinary sources were collected by employing a nonprobability sampling technique. Isolation and phenotypic identification of carbapenem-resistant K. pneumoniae were performed according to the CLSI standard. Molecular detection of various antibiotic resistance genes (ARGs) was done through PCR by using specific primers against each ARG. According to the pasture scheme, the multilocus sequence typing (MLST) was performed to characterize the K. pneumoniae sequence types (STs).Results: According to the results of the study, overall 9.4% (13/138) isolates were confirmed carbapenem-resistant K. pneumoniae. Among various carbapenem ARGs particularly, the blaNDM-1 was found in 92.3% (12/13) isolates followed by blaOXA-48 84.6% (11/13). MLST results revealed that overall 3 STs were found in the study which includes ST29, ST11, and ST258. Taking together, this is the first study to our best knowledge which demonstrated the prevalence of carbapenem-resistant K. pneumoniae and its various STs prevalent in veterinary settings and the waste of Pakistan.Conclusion: Based on the above-mentioned facts, we suggested that veterinary settings and waste are the potential source and reservoir of carbapenem-resistant K. pneumoniae, which may be disseminated to the environment and ultimately can affect the public and companion livestock health.Keywords: veterinary settings, carbapenem resistance, Klebsiella pneumoniae, Pakistan

Lubricants are used during the metal cutting process to remove the heat generated during the process and to reduce the Coefficient of friction (CoF). In this paper an attempt was made to synthesize an environment-friendly nano-lubricant using virgin coconut oil as base fluid by separately blending Copper (Cu) and Silver (Ag) nanoparticles in concentrations of 0.1%, 0.25%, 0.5% and 1% on the mass basis. The nanoparticles were blended in coconut oil by the means of ultrasonic shaking to obtain a uniform blend. The dependence of the Coefficient of friction of nanofluids and its performance on the concentration of the nanoparticles is well reported by researchers; therefore the optimal concentration of the nanoparticles was identified by Pin-on-Disk tribological testing. The following tests were conducted on the synthesised nano-lubricant such as Coefficient of friction (Pin-on-disk test), viscosity, flash & fire point and real time turning operation to determine the cutting force, and operating temperature. From the wear test results, it was inferred that copper added coconut nanofluids exhibited lower Coefficient of friction as compared to silver added coconut nanofluids. Further, it was identified that the lowest Coefficient of friction in the range was exhibited by copper added coconut nanofluids of 0.25% concentration. When these nanolubricants were tested in a real time turning operation using lathe tool dynamometer, it was found that 0.25% concentration of copper added coconut nanolubricant exhibited minimum cutting force and operating temperature indicating minimum coefficient of friction. Keywords: Nanolubricants, Cutting fluid, Cutting force, Cutting temperature, Coefficient of friction

Bilal Aslam,1 Wei Wang,2 Muhammad Imran Arshad,3 Mohsin Khurshid,1,4 Saima Muzammil,1 Muhammad Hidayat Rasool,1 Muhammad Atif Nisar,1 Ruman Farooq Alvi,1 Muhammad Aamir Aslam,2 Muhammad Usman Qamar,1 Muhammad Khalid Farooq Salamat,5 Zulqarnain Baloch6 1Department of Microbiology, Government College University Faisalabad, Faisalabad, Pakistan; 2NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing, China; 3Institute of Microbiology, University of Agriculture Faisalabad, Faisalabad, Pakistan; 4College of Allied Health Professionals, Directorate of Medical Sciences, Government College University Faisalabad, Faisalabad, Pakistan; 5Neurobiology Division, The Roslin Institute, University of Edinburgh, Edinburgh, UK; 6College of Veterinary Medicine, South China Agricultural University, Guangzhou, China Abstract: The advent of multidrug resistance among pathogenic bacteria is imperiling the worth of antibiotics, which have previously transformed medical sciences. The crisis of antimicrobial resistance has been ascribed to the misuse of these agents and due to unavailability of newer drugs attributable to exigent regulatory requirements and reduced financial inducements. Comprehensive efforts are needed to minimize the pace of resistance by studying emergent microorganisms, resistance mechanisms, and antimicrobial agents. Multidisciplinary approaches are required across health care settings as well as environment and agriculture sectors. Progressive alternate approaches including probiotics, antibodies, and vaccines have shown promising results in trials that suggest the role of these alternatives as preventive or adjunct therapies in future. Keywords: antibiotics, multidrug resistance, evolution, alternative therapies

Zulqarnain Baloch,1,* Bilal Aslam,2,* Saima Muzammil,2 Mohsin Khurshid,3 Muhammad Hidayat Rasool,2 Ke Ma4 1College of Veterinary Medicine, South China Agricultural University, Guangzhou, China; 2Department of Microbiology, Government College University Faisalabad, Faisalabad, Pakistan; 3College of Allied Health Professionals, Directorate of Medical Sciences, Government College University Faisalabad, Faisalabad, Pakistan; 4College of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, China *These authors contributed equally to this work Abstract: Antibiotic therapy has a dual impact: wanted, in which it immediately inhibits the growth of bacteria and the unwanted, which is responsible for the evolution of antibiotic resistance. The dissociation of therapeutic effectiveness from the possible risk of the antibiotic resistance may be attained by taking the advantage of specific relations between these drugs, and the methods in which mutations associated with resistance against a specific antibiotic may modify these relations or it may increase the sensitivity of the bacterium to the other antibiotics. Although the practical implementation of this notion needs considerable advancement and confirmation that depends upon the improvements in the field of genomics and diagnostics, these interventions propose new paradigms, which may confine or inverse the evolution of antibiotic resistance. Keywords: antibiotic resistance, inversion, mutations, bacteria, evolution

To guard consumers from meat adulteration, there is a dire obligation to encompass a sensitive, simple, and precise way for the identification of meat animal species. Molecular techniques have hoisted the expectations of developing authentic and reliable methods for testing the origin of meat species. This study explains the utilization of Polymerase Chain Reaction and Restriction Fragment Length Polymorphism along with mitochondrial cytochrome c oxidase subunit I gene to identify six meat species i.e., buffalo (Bubalus bubalis), cow (Bos indicus), cat (Felis catus), goat (Capra hircus), donkey (Equus asinus) and sheep (Ovis aries). Universal primers were utilized to amplify a specific region (approx. 710 bp) encoding the mitochondrial COI gene in each species and after insilico analysis two restriction enzymes (TasI and TruI) were selected to digest all desired amplicons. Restriction analysis on 3% agarose gel uncovered unique speciesspecific restriction profiles. The level of COI variation by utilizing TasI and TruI was found to be adequate to produce effectively analyzable species-specific profiles that could recognize all species unambiguously. This study suggested that PCR-RFLP is a swift and reliable scheme in favor of recognition and discrimination of analyzed meat species by mitochondrial COI gene and can be employed in food control laboratories. [ABSTRACT FROM AUTHOR]

The present work was designed to characterise novel indigenous strains of lactobacilli and to identify and evaluate their antagonistic activity against enteric pathogens. A total of four lactobacilli strains were isolated from Dahi samples (continental yogurt) and characterised using phenotypic and biochemical tests, and by 16S rDNA sequencing. The isolates were identified as Lactobacillus paracasei SABA3, L. paracasei SABA4, L. fermentum SABA5, and L. casei SABA6. The probiotic potential of these strains was assessed under the gastrointestinal simulating environment. The antagonistic activity of lactic acid bacilli (LAB) strains against multidrug-resistant (MDR) test pathogens was determined by in vitro assays. Moreover, the bactericidal activity of cell-free culture supernatant (CFCS) was determined by agar well diffusion and growth inhibition assays. All the LAB strains were tolerant to 0.3% bile salts, extreme acidic conditions (stomach environment) as well as pH > 6.5 (small intestine); and can efficiently grow at 37°C. Moreover, the isolates were found as non-haemolytic which confirms their safety and use as potential probiotics. The genetic screening has shown the presence of qnrS, tetK, tetW, vanR, vanX, and qnrA antibiotic-resistant determinants. The L. fermentum SABA5 exhibited the strongest bactericidal activity against the pathogenic bacterial species among all of these potential probiotic strains. The CFCS inhibited the proliferation of pathogenic bacterial species in a concentration-dependent manner. The chromatographic profiling of all isolates depicted lactic acid as a major fermentation product. [ABSTRACT FROM AUTHOR]

The emergence of antimicrobial resistance among biofilm forming pathogens aimed to search for the efficient and novel alternative strategies. Metallic nanoparticles have drawn a considerable attention because of their significant applications in various fields. Numerous methods are developed for the generation of these nanoparticles however, mycogenic (fungal-mediated) synthesis is attractive due to high yields, easier handling, eco-friendly and being energy efficient when compared with conventional physico-chemical methods. Moreover, mycogenic synthesis provides fungal derived biomolecules that coat the nanoparticles thus improving their stability. The process of mycogenic synthesis can be extracellular or intracellular depending on the fungal genera used and various factors such as temperature, pH, biomass concentration and cultivation time may influence the synthesis process. This review focuses on the synthesis of metallic nanoparticles by using fungal mycelium, mechanism of synthesis, factors affecting the mycosynthesis and also describes their potential applications as antioxidants and antibiofilm agents. Moreover, the utilization of mycogenic nanoparticles as quorum quenching agent in hampering the bacterial cell-cell communication (quorum sensing) has also been discussed., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Cadmium (Cd) is a highly toxic metal that frequently contaminates our environment. In this study, the bioflocculant-producing, cadmium-resistant Escherichia fergusonii ZSF-15 was characterized from Paharang drain, Bawa Chak, Faisalabad, Pakistan. The Cd-resistant E. fergusonii was used to determine the bioflocculant production using yeast-peptone-glycerol medium (pH 6.5) supplemented with 50 mg L -1 of Cd. The culture was incubated for 3 days at 37 °C in a rotary shaker at 120 rpm. The fermentation broth was centrifuged at 4000 g for 10 min after the incubation period. The maximum flocculating activity by isolate ZSF-15 was found to be 71.4% after 48 h of incubation. According to the Fourier transform infrared spectroscopy analysis, the bioflocculant produced by strain ZSF-15 was comprised of typical polysaccharide and protein, i.e. hydroxyl, carboxyl, and amino groups. The strain ZSF-15 exhibited bioflocculant activity at range of pH (6-8) and temperature (35-50℃). Maximum flocculation activity (i.e. 71%) was observed at 47℃, whereas 63% flocculation production was observed at pH 8. In the present study, antioxidant enzyme profile of ZSF-15 was also evaluated under cadmium stress. A significant increase in antioxidant enzymes including superoxide dismutase (118%) and ascorbate peroxidase (28%) was observed, whereas contents of catalase (86%), glutathione transferase (13%), and peroxidase (8%) were decreased as compared to control., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Chromium (Cr) is classified as a toxic metal as it exerts harmful effects on plants and human life. Bacterial-assisted nano-phytoremediation is an emerging and environment friendly technique that can be used for the detoxification of such pollutants. In current study, pot experiment was conducted in which spinach plants were grown in soil containing chromium (0, 5, 10, 20 mgkg -1 ) and treated with selected strain of Bacillus sp. and Cu-O nanoparticle (CuONPs). Data related to plant's growth, physiological parameters, and biochemical tests was collected and analyzed using an appropriate statistical test. It was observed that under chromium stress, all plant's growth parameters were significantly enhanced in response to co-application of CuONPs and Bacillus sp. Similarly, higher levels of catalase, superoxide dismutase, malondialdehyde, and hydrogen peroxide were also observed. However, contents of anthocyanin, carotenoid, total chlorophyll, chlorophyll a & b, were lowered under chromium stress, which were raised in response to the combined application of CuONPs and Bacillus sp. Moreover, this co-application has significant positive effect on total soluble protein, free amino acid, and total phenolics. From this study, it was evident that combined application of Bacillus sp. and CuONP alleviated metal-induced toxicity in spinach plants. The findings from current study may provide new insights for agronomic research for the utilization of bacterial-assisted nano-phytoremediation of contaminated sites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Phytonanotechnology plays a crucial part in the production of good quality and high-yield food. It can also alter the plant's production systems, hence permitting the efficient, controlled and stable release of agrochemicals such as fertilizers and pesticides. An advanced understanding of nanomaterials interaction with plant responses like localization and uptake, etc. could transfigure the production of crops with high disease resistance and efficient nutrients utilization. In agriculture, the use of nanomaterials has gained acceptance due to their wide-range applications. However, their toxicity and bioavailability are the major hurdles for their massive employment. Undoubtedly, nanoparticles positively influence seeds germination, growth and development, stress management and post-harvest handling of vegetables and fruits. These nanoparticles may also cause toxicity in plants through oxidative stress by generation of excessive reactive oxygen species thus affecting the cellular biomolecules and targeting different channels. Nanoparticles have shown to exert various effects on plants that are mainly affected by various attributes such as physicochemical features of nanomaterials, coating materials for nanoparticles, type of plant, growth stages and growth medium for plants. This article discusses the interaction, accretion and toxicity of nanomaterials in plants. The factors inducing nanotoxicity and the mechanisms followed by nanomaterials causing toxicity are also instructed. At the end, detoxification mechanism of plant is also presented., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Since long, medicinal plants or herbs are being used in different traditional treatment systems as therapeutic agents to treat a variety of illnesses. Bixa orellana L., an medicinal plant (family: Bixaceae), is an Ayurvedic herb used to treat dyslipidemia, diarrhoea, and hepatitis since ancient times. B. orellana L., seeds contain an orange-red coloured component known as bixin (C 25 H 30 O 4 ), which constitutes 80% of the extract.Chemically, bixin is a natural apocarotenoid, biosynthesized through the oxidative degradation of C40 carotenoids. Bixin helps to regulate the Nrf2/MyD88/TLR4 and TGF-1/PPAR-/Smad3 pathways, which further give it antifibrosis, antioxidant, and anti-inflammatory properties. This current review article presents a comprehensive review of bixin as an anti-inflammatory, antioxidant, anticancer,and skin protecting natural product. In addition, the biosynthesis and molecular target of bixin, along with bixin extraction techniques, are also presented., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Biopolymer based metal oxide nanoparticles, prepared by eco-friendly approach, are gaining interest owing to their wide range of applications. In this study, aqueous extract of Trianthema portulacastrum was used for the green synthesis of chitosan base copper oxide (CH-CuO) nanoparticles. The nanoparticles were characterized through UV-Vis Spectrophotometry, SEM, TEM, FTIR and XRD analysis. These techniques provided evidence for the successful synthesis of the nanoparticles, having poly-dispersed spherical shaped morphology with average crystallite size of 17.37 nm. The antibacterial activity for the CH-CuO nanoparticles was determined against multi-drug resistant (MDR), Escherichia coli, Pseudomonas aeruginosa (gram-negative), Enterococcus faecium and Staphylococcus aureus (gram-positive). Maximum activity was obtained against Escherichia coli (24 ± 1.99 mm) while least activity was observed against Staphylococcus aureus (17 ± 1.54 mm). In-vitro analysis for biofilm inhibition, EPS and cell surface hydrophobicity showed >60 % inhibitions for all the bacterial isolates. Antioxidant and photocatalytic assays for the nanoparticles showed significant activities of radical scavenging (81 ± 4.32 %) and dye degradation (88 %), respectively. Antidiabetic activity for the nanoparticles, determined by in-vitro analysis of alpha amylase inhibition, showed enzyme inhibition of 47 ± 3.29 %. The study signifies the potential of CH-CuO nanoparticle as an effective antimicrobial agent against MDR bacteria along with the antidiabetic and photocatalytic activities., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to provide a comparative analysis of chitosan (CH), copper oxide (CuO), and chitosan-based copper oxide (CH-CuO) nanoparticles for their application in the healthcare sector. The nanoparticles were synthesized by a green approach using the extract of Trianthema portulacastrum . The synthesized nanoparticles were characterized using different techniques, such as the synthesis of the particles, which was confirmed by UV-visible spectrometry that showed absorbance at 300 nm, 255 nm, and 275 nm for the CH, CuO, and CH-CuO nanoparticles, respectively. The spherical morphology of the nanoparticles and the presence of active functional groups was validated by SEM, TEM, and FTIR analysis. The crystalline nature of the particles was verified by XRD spectrum, and the average crystallite sizes of 33.54 nm, 20.13 nm, and 24.14 nm were obtained, respectively. The characterized nanoparticles were evaluated for their in vitro antibacterial and antibiofilm potential against Acinetobacter baumannii isolates, where potent activities were exhibited by the nanoparticles. The bioassay for antioxidant activity also confirmed DPPH scavenging activity for all the nanoparticles. This study also evaluated anticancer activities of the CH, CuO, and CH-CuO nanoparticles against HepG2 cell lines, where maximum inhibitions of 54, 75, and 84% were recorded, respectively. The anticancer activity was also confirmed by phase contrast microscopy, where the treated cells exhibited deformed morphologies. This study demonstrates the potential of the CH-CuO nanoparticle as an effective antibacterial agent, having with its antibiofilm activity, and in cancer treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sarfraz, Zubair, Aslam, Ashraf, Siddique, Hayat, Cruz, Muzammil, Khurshid, Sarfraz, Hashem, Dawoud, Avila-Quezada and Abd_Allah.)

As is well known, plant products have been increasingly utilized in the pharmaceutical industry in recent years. By combining conventional techniques and modern methodology, the future of phytomedicines appears promising. Pogostemon Cablin (patchouli) is an important herb used frequently in the fragrance industries and has various therapeutic benefits. Traditional medicine has long used the essential oil of patchouli ( P. cablin ) as a flavoring agent recognized by the FDA. This is a gold mine for battling pathogens in China and India. In recent years, this plant has seen a significant surge in use, and approximately 90% of the world's patchouli oil is produced by Indonesia. In traditional therapies, it is used for the treatment of colds, fever, vomiting, headaches, and stomachaches. Patchouli oil is used in curing many diseases and in aromatherapy to treat depression and stress, soothe nerves, regulate appetite, and enhance sexual attraction. More than 140 substances, including alcohols, terpenoids, flavonoids, organic acids, phytosterols, lignins, aldehydes, alkaloids, and glycosides, have been identified in P. cablin . Pachypodol (C18H16O7) is an important bioactive compound found in P. cablin. Pachypodol (C18H16O7) and many other biologically essential chemicals have been separated from the leaves of P. cablin and many other medicinally significant plants using repeated column chromatography on silica gel. Pachypodol's bioactive potential has been shown by a variety of assays and methodologies. It has been found to have a number of biological activities, including anti-inflammatory, antioxidant, anti-mutagenic, antimicrobial, antidepressant, anticancer, antiemetic, antiviral, and cytotoxic ones. The current study, which is based on the currently available scientific literature, intends to close the knowledge gap regarding the pharmacological effects of patchouli essential oil and pachypodol, a key bioactive molecule found in this plant.