Alpha toxin production potential and antibiotic resistance patterns of clostridium perfringens isolates from meat samples.

Objective: This research aimed to analyze the prevalence, molecular characteristics, toxinotyping, alpha toxin production potential, and antibiotic resistance pattern of Clostridium perfringens (C. perfringens) isolates in meat samples collected from various sources. Methods: Sixty meat samples were screened for alpha toxin using Enzyme-Linked Immunosorbent Assay (ELISA), revealing a positivity rate of 13.3%, predominantly in raw poultry meat. Subsequent culturing on Perfringens agar identified nine samples harboring characteristic C. perfringens colonies, primarily isolated from raw poultry meat. Molecular confirmation through 16S rRNA gene amplification and sequencing authenticated twelve isolates as C. perfringens, with nine strains exhibiting genetic resemblance to locally isolated strains. Toxinotyping assays targeting alpha toxin-specific genes confirmed all nine isolates as type A C. perfringens, with no detection of beta or epsilon toxin genes. Hemolytic assays demonstrated varying alpha toxin production potentials among isolates, with accession number OQ721004.1 displaying the highest production capacity. Moreover, antibiotic resistance profiling revealed multi-drug resistance patterns among the isolates. Results: The study identified distinct clusters within C. perfringens strains, indicating variations. Phylogenetic analysis delineated genetic relatedness among strains, elucidating potential evolutionary paths and divergences. Conclusion: The findings underscore the need for robust surveillance and control measures to mitigate the risk of C. perfringens contamination in meat products, particularly in raw poultry meat. Enhanced monitoring and prudent antimicrobial stewardship practices are warranted in both veterinary and clinical settings to address the observed antibiotic resistance profiles and prevent foodborne outbreaks.

The Role of Soil Abundance of TxtAB in Potato Common Scab Disease Severity.

Common scab is an economically costly soilborne disease of potato endemic in many potato-growing regions. The disease is caused by species of Streptomyces bacteria that produce the phytotoxin thaxtomin A. The primary disease management tool available to growers is planting resistant cultivars, but no cultivar is fully resistant to common scab, and partially resistant cultivars are often not the preferred choice of growers because of agronomic or market considerations. Therefore, growers would benefit from knowledge of the presence and severity of common scab infestations in field soils to make informed planting decisions. We implemented a quantitative PCR diagnostic assay to enable field detection and quantification of all strains of Streptomyces that cause common scab in the United States through amplification of thaxtomin A biosynthetic genes. Greenhouse trials confirmed that pathogen abundance was highly correlated with disease severity for five distinct phytopathogenic Streptomyces species, although the degree of disease severity was dependent on the pathogen species. Correlations between the abundance of the thaxtomin biosynthetic genes from field soil with disease on tubers at field sites across four U.S. states and across 2 years were not as strong as correlations observed in greenhouse assays. We also developed an effective droplet digital PCR diagnostic assay that also has potential for field quantification of thaxtomin biosynthetic genes. Further improvement of the PCR assays and added modeling of other environmental factors that impact disease outcome, such as soil composition, can aid growers in making informed planting decisions.

Methanol-based biomanufacturing of fuels and chemicals using native and synthetic methylotrophs.

Methanol has recently gained significant attention as a potential carbon substrate for the production of fuels and chemicals, owing to its high degree of reduction, abundance, and low price. Native methylotrophic yeasts and bacteria have been investigated for the production of fuels and chemicals. Alternatively, synthetic methylotrophic strains are also being developed by reconstructing methanol utilization pathways in model microorganisms, such as Escherichia coli. Owing to the complex metabolic pathways, limited availability of genetic tools, and methanol/formaldehyde toxicity, the high-level production of target products for industrial applications are still under development to satisfy commercial feasibility. This article reviews the production of biofuels and chemicals by native and synthetic methylotrophic microorganisms. It also highlights the advantages and limitations of both types of methylotrophs and provides an overview of ways to improve their efficiency for the production of fuels and chemicals from methanol.

First Report of Leaf Spot of Sugar Beet Caused by Stemphylium vesicarium in Minnesota, USA.

In July 2021, sugar beet (Beta vulgaris L.) leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected from a field in Minnesota (MN) (46.2774° N, 96.3100° W), with 15% disease incidence and 30% disease severity. Leaves were washed with tap water then surface disinfected in 1% NaOCl aqueous solution for 1 min. Samples were rinsed thrice with sterile distilled water and dried in a laminar flow hood. A 2-cm leaf disc was plated on potato dextrose agar amended with streptomycin sulfate (200 mg/L) and incubated for four days at 25°C under 12-h light/dark cycle. Single spore cultures were obtained by suspending in sterile water spores harvested from a single colony. The suspension was streaked on a dish with V8 agar media and incubated as described. Five pure cultures were transferred to clarified V8 agar media for morphological feature observations. Colonies were uniform in appearance and developed light to olivaceous green mycelium. Conidia were dark brown to olivaceous green in color and measured 30 × 18 µm (n=20). They were oblong to broadly oval shaped muriform, and multiseptated (1 to 5 septa). Hyphae were septate and pale brown. Conidiophores were short, septate, and light to dark brown in color. Based on the morphological characteristics, isolates were identified as Stemphylium vesicarium (Simmons 1969). Genomic DNA of all five isolates were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR amplification and sequencing of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers), the largest subunit of RNA polymerase II (5F2/7cR primers) (O'Donnell et al. 2009), the plasma membrane ATPase (ATPD-F1/ATPD-R1) gene (Lawrence et al. 2013), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) (gpd1/gpd2) (Berbee et al. 1999), and ß-tubulin gene (Bt2a/Bt2b primers) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (ß-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (ß-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants. S. vesicarium spore suspensions (1 × 106/ml) were sprayed on three leaves from each plant. This trial was repeated with three replicates. A similar group of plants were sprayed with autoclaved distilled water to serve as non-inoculated control. All plants were incubated in the mist chamber for 5 days at 25°C, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Fifteen days post-inoculation, all inoculated plants had multiple lesions with dark brown margins with a grayish center, and non-inoculated control plants were asymptomatic. The re-isolated fungus was morphologically similar to isolates retrieved from the field. S. vesicarium was reported on sugar beet in Michigan (Metheny et al. 2022). This is the first report of S. vesicarium causing disease on sugar beet in MN. Stemphylium sp. is a major problem of sugar beet in the Netherlands (Hanse et al. 2015). Efforts should be made to prevent introduction of susceptible beet cultivars so that the disease does not become widespread in the USA.

Evaluation of Antimicrobial Effect of Silver Nanoparticle Based Whey Emulsions and Edible Films for the Extension of Shelf Life of Fruits and Vegetables.

The purpose of this study was to develop and assess the antimicrobial properties of silver nanoparticles (AgNPs)-based whey emulsions and edible films for extending the shelf life of fruits and vegetables. The AgNPs were synthesized using a biological method, and their morphological and topographical characteristics were evaluated using scanning electron microscopy (SEM). The AgNPs were incorporated into the emulsions and films to increase their antimicrobial efficacy. Bacterial and fungal strains were identified by DNA regions, including 16S and 18S rRNA, TEF-1α, and RPB2 to evaluate antimicrobial activity. AgNPs-based emulsions and films were used to extend the shelf life of fruits and vegetables for up to 15 days. The results showed that the use of AgNPs in the coated samples significantly increased their effectiveness against bacterial and fungal strains. SEM analysis revealed the presence of AgNPs of varying sizes, ranging from 21 to 62 nm. The zones of inhibition were measured against Staphylococcus aureus, Escherichia coli, Salmonella enterica, Aspergillus flavus, Aspergillus tamari, and Aspergillus niger. The total viable count (log cfu/ml) decreased from 6.423 in the control group to 3.301 in the treated samples. The antioxidant activity of the treated fruits and vegetables was also significantly improved, with values of 56.12, 23.36, 26.10, 7.6, 36.04, and 33.81% for strawberry, taro root, guava, peas, green chili, and carrot, respectively (p < 0.05). The AgNPs-based whey protein emulsions were found to exhibit the highest antimicrobial activity and are therefore a promising approach to extend the shelf life of fruits and vegetables.

Reconstructing ethanol oxidation pathway in Pseudomonas putida KT2440 for bio-upgrading of ethanol to biodegradable polyhydroxybutanoates.

Ethanol has recently been demonstrated as a suitable carbon source for acetyl-CoA-derived products with high theoretical yield. Herein, the short-chain-length polyhydroxyalkanoates production pathway was constructed in an industrial platform P. putida KT2440, allowing the engineered strain to produce 674.97 ± 22.3 mg/L of Polyhydroxybutyrate (PHB) from ethanol as sole carbon source. Furthermore, the ethanol catabolic pathway was reconstructed to enhance the acetyl-coA pool by expressing the novel Aldehyde dehydrogenases from Klebsiella pneumonia and Dickeya zeae, resulting in a titer of 1385.34 ± 16.5 mg/L and 9300 ± 0.56 mg/L of PHB in shake flask and fermenter, respectively. Furthermore, transcriptome analysis was conducted to provide insights into the central metabolic pathways and different expression patterns in response to changes in substrate. Additionally, the production of co-polymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) was shown using glycerol and ethanol as co-substrates from recombinant P. putida KT2440. This work demonstrates the potential of P. putida KT2440 as a promising industrial platform for short-chain-length PHAs production from structurally unrelated carbon sources.

Activity of plant essential oils against antibiotic resistant Enterococcus faecalis isolated from diarrheic children.

Activity of plant essential oils and their fractions was evaluated against characterized isolates of antibiotic resistant Enterococcus faecalis recovered from diarrheic children. The isolates were confirmed by polymerase chain reaction (PCR) targeting 16S rRNA gene amplification followed by nucleotide sequencing and accession numbers retrieved were MW349990.1, MW349859.1, MW332122.1, MW356805.1, MW349975.1, MW349988.1, MW356790.1, MW356244.1, MW341593.1 and MW332549.1. These isolates were screened for antibiotic susceptibility to a wide range of antibiotic groups and mean zone of inhibition (ZOI) of all antibiotics were recorded. Antibacterial activity of plant essential oils (n=05) was checked against three antibiotic resistant isolates of E. faecalis. Three plant essential oils having higher ZOI including Cinnamomum verum, Syzygium aromaticum and Nigella sativa were used against resistant E. faecalis isolates to determine minimum inhibitory concentration (MIC). The lowest MIC observed was of S. aromaticum (11.39±3.94 mg mL-1). The S. aromaticum n-hexane plus chloroform fraction displayed higher mean ZOI (16.67±2.51 mm), while the lowest MIC was of n-hexane oil fraction. Based upon gas chromatography-mass spectrometry (GC/MS) analysis, the most effective fatty acid was eugenic acid which is present in higher proportion in both fractions. These fractions of essential oils proved safe for the treatment of antibiotic resistant diarrheic cases of children caused by E. faecalis.

In vitro and in silico Studies Reveal Bacillus cereus AA-18 as a Potential Candidate for Bioremediation of Mercury-Contaminated Wastewater.

Mercury (Hg) pollution is a worldwide problem and increasing day by day due to natural and anthropogenic sources. In this study, mercury-resistant (HgR) bacterial isolates were isolated from industrial wastewater of Ittehad Chemicals Ltd., Kala Shah Kaku, Lahore, Pakistan. Out of 65 bacterial isolates, five isolates were screened out based on showing resistance at 30-40 µg/ml against HgCl2. Selected Hg-resistant bacterial isolates were characterized as Bacillus subtilis AA-16 (OK562835), Bacillus cereus AA-18 (OK562834), Bacillus sp. AA-20 (OK562833), Bacillus paramycoides AA-30 (OK562836), and Bacillus thuringiensis AA-35 (OK562837). B. cereus AA-18 showed promising results in the resistance of HgCl2 (40 µg/ml) due to the presence of merA gene. Scanning electron microscopy (SEM) analysis of immobilized B. cereus AA-18 showed the accumulation Hg on the cell surface. The inoculation of immobilized B. cereus AA-18 remediated 86% Hg of industrial wastewater up to 72 h at large scale (p < 0.05). In silico analysis showed structural determination of MerA protein encoded by merA gene of B. cereus AA-18 (OK562598) using ProtParam, Pfam, ConSurf Server, InterPro, STRING, Jpred4, PSIPRED, I-TASSER, COACH server, TrRosetta, ERRAT, VERIFY3D, Ramachandran plot, and AutoDock Vina (PyRx 8.0). These bioinformatics tools predicted the structural-based functional homology of MerA protein (mercuric reductase) associated with mer operon harboring bacteria involved in Hg-bioremediation system.

Molecular markers for the detection of pathogenic and food poisoning potential of methicillin resistant Staphylococcus aureus isolated from wounds of hospitalized patients.

Pathogenic strains of Staphylococcus aureus are mostly resistant to methicillin and they can cause severe infections. The current study was planned to assess the food poisoning potential of pathogenic, methicillin resistant Staphylococcus aureus by molecular detection of enterotoxin A (Eta) gene. A total of 100 septic wound samples from patients admitted in surgical ward (n=50) and burn unit (n=50) of Mayo Hospital Lahore were collected aseptically. These samples were processed primarily for bacterial growth on nutrient agar and purified on mannitol salt agar where twenty (20) samples showed pin-point colonies with yellow discoloration of media. Moreover, isolates were further characterized on the basis of microscopic appearance and biochemical assays where fourteen (14) isolates were declared Staphylococcus. DNA of these isolates were subjected to 16S rRNA gene amplification and sequences of S. aureus were submitted to NCBI GenBank viz., MW344063.1, MW341438.1, MW344064.1, MW344065.1, MW341439.1, MW341440.1, MW345971.1, MW345972.1, MW345973.1, MW716458.1. All the isolates (n=10) demonstrated molecular confirmation of pathogenicity and methicillin resistance by amplification of Coa and mecA gene. Out of these ten isolates, three amplified enterotoxin A (Eta) gene were confirmed. It is concluded that enterotoxin A of S. aureus which causes food poisoning is present in pathogenic, methicillin resistant S. aureus isolated from various wounds infections.

Bio-upgrading of ethanol to fatty acid ethyl esters by metabolic engineering of Pseudomonas putida KT2440.

Fatty acid ethyl esters (FAEEs) have gained increasing attention as a replacement for traditional fossil fuels in the recent years. Here, we report the efficient upgrading of ethanol to FAEEs from Pseudomonas putida KT2440, using ethanol as the sole carbon source. First, the wax synthase (WS) encoded by the atfA gene from Acinetobacter baylyi ADP1 was expressed in P. putida KT2440. Second, the flux from ethanol towards acetyl-CoA was increased by expression of the acetaldehyde dehydrogenase (ada) from Dickeya zeae. By using dodecane overlay to capture FAEEs, 1.2 g/L of FAEEs with a yield of 152.09 mg FAEEs/g ethanol were produced. Culture optimization enhanced the FAEEs contents up to 1.6 g/L in shake flask and 4.3 g/L in a fed-batch fermenter. In summary, our study provides a basis for combining the bioethanol production process with the efficient upgrading of ethanol to biodiesel.

Antibiotyping and genotyping of extensively drug-resistant (XDR) Salmonella sp. isolated from clinical samples of Lahore, Pakistan.

AIMS: Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi. The objective of this study was to evaluate the prevalence of XDR Salmonella among local population of Lahore and genotyping of isolates for antibiotic-resistant genes. METHODS AND RESULTS: A total of 200 blood samples from suspected typhoid fever patients were collected. One hundred and fifty-seven bacterial samples were confirmed as Salmonella Typhi and 23 samples were confirmed as Salmonella Paratyphi after biochemical, serological and PCR based molecular characterization. Antibiogram analysis classified 121 (67·2%) Salmonella isolates as MDR and 62 isolates (34·4%) as XDR. The predominant resistance gene was ampC with 47·7% prevalence, followed by gyrA, catA1, tet(A), aac (3)-la, qnrS, blaNDM-1 and blaCTX-M-15 genes in 45·5, 40, 21·6, 18·3, 11·6, 2·2 and 0·5% isolates respectively. Sequence analysis showed the presence of sul1 and dfrA7 gene cassette arrays in 12 class 1 integron integrase positive isolates. CONCLUSION: Large number of clinical XDR S. Typhi-resistant against third generation cephalosporins have been reported. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study highlights the possible emergence of clinical XDR S. Typhi cases in Lahore, Pakistan. Potential attribution of phenotypic and genotypic XDR cases may help to contribute targeted therapy.

Characterization of Mercury-Resistant Rhizobacteria for Plant Growth Promotion: An In Vitro and In Silico Approach.

In this study, a total 30 rhizobacterial isolates were screened out based on resistance against different concentrations of mercuric chloride (HgCl2), growth on nitrogen-free mannitol (NFM) and production of indole-3-acetic acid (IAA). The biochemical and plant growth promoting characterization of selected isolates was performed by different biochemical tests. Out of 30, six isolates, UM-3, AZ-5, UM-7, UM-11, UM-26, and UM-28 showed resistance at 30 µg/ml HgCl2, pronounced growth on NFM and high production of IAA as 18.6, 16.7, 16, 18.7, 14, and 16 µg/ml, respectively (P < 0.05). The 16S rDNA ribotyping and phylogenetic analysis of selected bacterial isolates were performed and characterized as Exiguobacterium sp. UM-3 (KJ736011), Bacillus thuringiensis AZ-5 (KJ675627), Bacillus subtilis UM-7 (KJ736013), Enterobacter cloacae UM-11 (KJ736014), Pseudomonas aeruginosa UM-26 (KJ736016), P. aeruginosa UM-28 (KJ736017) and Bacillus pumilus UM-16 (KJ736015) used as negative control. B. thuringiensis AZ-5 showed high resistance against 30 µg/ml of HgCl2 due to the presence of merB gene. The structural determination of MerB protein was carried out using bioinformatics tools, i.e., Protparam, Pfam, InterProScan, STRING, Jpred4, PSIPRED, I-TASSER, COACH server and ERRAT. These tools predicted the structural based functional homology of MerB protein (organomercuric lyase) in association with MerA (mercuric reductase) in bacterial Hg-detoxification system.

A Potential Biocontrol Agent Streptomyces violaceusniger AC12AB for Managing Potato Common Scab.

Potato common scab (PCS) is an economically important disease worldwide. In this study we demonstrated the possible role of Streptomyces violaceusniger AC12AB in controlling PCS. Isolates of Streptomyces scabies were obtained from CS infected tubers collected from Maine United States, which were confirmed by morphological and molecular analysis including 16S rRNA sequencing and RFLP analysis of amplified 16S-23S ITS. Pathogenicity assays related genes including txtAB, nec1, and tomA were also identified in all S. scabies strains through PCR reaction. An antagonistic bacterial strain was isolated from soil in Punjab and identified as S. violaceusniger AC12AB based on 16S rRNA sequencing analysis. Methanolic extract of S. violaceusniger AC12AB contained azalomycin RS-22A which was confirmed by 1H and 13C-NMR, 1H/1H-COSY, HMBC and HMQC techniques. S. violaceusniger AC12AB exhibited plant growth promotion attributes including Indole-3-acetic acid production with 17 µgmL-1 titers, siderophores production, nitrogen fixation and phosphates solubilization potential. When tubers were inoculated with S. violaceusniger AC12AB, significant (P < 0.05) PCS disease reduction up to 90% was observed in greenhouse and field trials, respectively. Likewise, S. violaceusniger AC12AB significantly (P < 0.05) increased potato crop up to 26.8% in field trial. Therefore, plant growth promoting S. violaceusniger AC12AB could provide a dual benefit by decreasing PCS disease severity and increasing potato yield as an effective and inexpensive alternative strategy to manage this disease.

Expression and Purification of Transmembrane Protein MerE from Mercury-Resistant Bacillus cereus.

Mercury-resistant (HgR) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to 40 µg/ml against mercuric chloride (HgCl2). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51- 100% homology with the corresponding region of the merA gene of already reported mercuryresistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury (Hg0) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing 30 µg/ml of HgCl2 was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

Biological Control of Potato Common Scab With Rare Isatropolone C Compound Produced by Plant Growth Promoting Streptomyces A1RT.

Potato is prone to many drastic diseases like potato common scab (CS). As no highly effective methods exist for managing CS, this study explored the possibility of using biological control. Ten bacterial strains were isolated from CS-infected potato tubers from four different locations of Punjab, Pakistan, and identified based on biochemical and molecular analysis. Analysis of 16s rDNA sequences amplified by PCR revealed the isolated bacterial strains to be Streptomyces scabies, S. turgidiscabies and S. stelliscabiei. Pathogenic islands were also confirmed among the isolates after identification of txtAB, nec1, and tomA genes with PCR amplification. One strain isolated from soil was antagonistic to the pathogenic Streptomyces spp., and determined to be Streptomyces A1RT on the basis of 16s rRNA sequencing. A methanolic extract of Streptomyces A1RT contained Isatropolone C, which was purified and structurally determined by 1H- and 13C-NMR, 1H/1H-COSY, HMQC, and HMBC techniques. Streptomyces A1RT also produced the plant growth hormone indole-3-acetic acid (IAA) with a titer of 26 µg ml-1 as confirmed by spectrophotometry and HPLC. In a greenhouse assay, disease severity index was established from 0 to 500. Average disease severity indexes were recorded as 63, 130.5, and 78 for Streptomyces scabies, S. turgidiscabies and S. stelliscabiei, respectively. When Streptomyces A1RT was applied in soil that contained one of these pathogenic isolates, the average disease severity indexes were significantly (P < 0.05) reduced to 11.1, 5.6 and 8.4, respectively. A significant increase in tuber weight and shoot development was also observed with the tubers treated with Streptomyces A1RT. The use of the plant growth-promoting Streptomyces A1RT against potato CS thus provides an alternative strategy to control the disease without affecting environmental, plants, animals and human health.

GC-MS characterisation and antibacterial activity evaluation of Nigella sativa oil against diverse strains of Salmonella.

Salmonella resistance is becoming a worldwide serious health issue in these days; therefore, it is an urgent need to develop some alternative approaches to overcome this problem. Twenty bacterial strains were isolated and purified from different environmental sources and confirmed as Salmonella by morphological and biochemical analyses. Further confirmation was done by 16s rRNA sequencing. Antibiotic susceptibility test was performed by well diffusion assay against different concentrations of Ceftriaxone and Ciprofloxacin. The behaviour of both antibiotics was different against diverse strains of Salmonella. Salmonella strains resistant to both antibiotics were analysed for antibacterial activity of natural extracts of Nigella sativa (black seeds). N. sativa oil was found to be more effective against Salmonella species for which even Ceftriaxone and Ciprofloxacin were ineffective. Gas chromatography and mass spectrometry analysis of N. sativa oil was also accomplished, exhibiting 10 compounds including thymoquinone, p-cymene, cis-carveol, thymol, α-phellandrene, α-pinene, ß-pinene, trans-anethole, α-longipinene and longifolene.