Insights into the microbiological and virulence characteristics of bacteria in orthopaedic implant infections: A study from Pakistan.

The exponential increase in the prevalence of multidrug resistant bacteria has resulted in limiting surgical treatment options globally, potentially causing biofilm-related complications, implant failure, and severe consequences. This study aims to isolate and characterize bacteria from post-surgical orthopaedic implant infections and screening for multiple antibiotic resistance. A cross-sectional study was conducted, involving isolation of forty-four dominant pathogenic bacterial isolates from 16 infected implant samples from across Islamabad and Rawalpindi. Out of forty-four, 38% cocci and 61% bacilli were obtained. Approximately 90% of isolates showed multiple antibiotic resistance (MAR) index of more than 0.2. Eleven strains were identified via 16S rRNA gene sequencing as Pseudomonas aeruginosa, Bacillus spp., Planococcus chinensis, Staphylococcus, Escherichia coli and Enterobacter cloacae. The bacterial strain E. coli MB641 showed sensitivity to Polymyxin only, and was resistant to all other antibiotics used. Maximum biofilm forming ability 0.532 ± 0.06, 0.55 ± 0.01 and 0.557 ± 0.07 was observed in Pseudomonas aeruginosa MB663, Pseudomonas aeruginosa MB664 and Bacillus spp. MB647 respectively after 24 hours of incubation. EPS production of bacterial strains was assessed, the polysaccharides and protein content of EPS were found to be in the range of 11-32 µg/ml and 2-10 µg/ml, respectively. Fourier transform infrared spectroscopic analysis of EPS showed the presence of carbohydrates, proteins, alkyl halides, and nucleic acids. X-ray diffraction analysis revealed crystalline structure of EPS extracted from biofilm forming bacteria. These findings suggest a high prevalence of antibiotic-resistant bacteria in orthopaedic implant-associated surgeries, highlighting the urgent need for ongoing monitoring and microorganism testing in infected implants.

Optimization of amylase production using response surface methodology from newly isolated thermophilic bacteria.

Present study was aimed at screening and characterizing thermostable amylase-producing bacteria from water and sediment samples of unexplored hot spring of Tatta Pani Kotli Azad Kashmir. Four thermophilic isolates were characterized on morphological, biochemical, physiological basis and were authenticated by molecular analysis. By 16S rDNA sequencing, isolates were identified as Anoxybacillus mongoliensis (MBT001), Anoxybacillus flavithermus (MBT002), Bacillus (MBT004). Among all identified strains, MBT003 showed maximum homology with both Anoxybacillus mongoliensis and Anoxybacillus flavithermus. Amylase activity was analyzed qualitatively in starch agar and quantitatively by DNS method. The optimal enzyme production was observed and authenticated by Response Surface Methodology at 7 pH, 70 °C, 1.25% substrate concentration, 300 µL of inocula volume after 48 h of incubation. Optimum amylase activity (4.4 U/mL) and stability (3.3 U/mL) was observed with 1.5% soluble starch at 70 °C. Maximum activity (3.7 U/mL) and stability (1.5 U/mL) was found at pH 8. Enzyme activity was increased in the presence of MgSO4 and CaCl2. Amylase was stable with surfactants and commercial detergents for 30 min. Supplementation of the enzyme with commercial detergent improved the washing ability of the detergent. This investigation has revealed that these thermostable bacteria are excellent source of amylase which can be used commercially for generating economic activity on sustainable basis.

Diversity of culturable thermophilic bacteria from Tata Pani hotspring of Kotli Azad Jammu and Kashmir.

Hot water springs are unique areas populated by mesophiles, thermotolerant and hyperthermophiles. They are the source of diversity of thermophiles, mainly belonging to archaea and bacteria domains. The diversity of thermophiles gives an outline of the huge biological potential that can be exploited for industrial applications.To this end, this study was aimed to isolate and characterise the unexplored thermophilic microorganisms from hot water spring in Tatapani, Tehsil & District Kotli AJK, Pakistan. Around 10 bacterial isolates were identified using morphological, biochemical, physiological and molecular attributes. Sequencing of the 16S rDNA gene of the isolates followed by BLAST search revealed that the strain MBT008 has 100% similarity with Anoxybacilluskamchatkensis. MBT012 showed 99.57% similarity with A.mongoliensis, MBT014 was affiliated with A.tengchongensis with 99.43% similarity, MBT009 showed 99.83% homology with A.gonensis and MBT018, 98.70% similarity with A.karvacharensis. The presence of all this microbial diversity in one common source is of immense importance related to envioronmental and industrial aspects in general and extraction of thermostable enzymes from these thermophiles specifically opens new horizons in the field of industrial biotechnology. These thermophiles are revealing new capabilities and are being manipulated by biotechnologists in utilizing them in different unique ways.

Metabolites produced by inoculated Vigna radiata during bacterial assisted phytoremediation of Pb, Ni and Cr polluted soil.

Phytoremediation assisted with plant growth promoting bacteria (PGPB) is a green technology to remediate metal contaminated soils. Plants usually produce secondary metabolites to tolerate metal toxicity. Present study was designed to explore the phytoremediation potential of Vigna radiata var. NM-II in the presence of metal resistant PGPB and comparison of metabolites produced under heavy metal stresses (Pb, Ni, Cr). Three PGPB selected for present study include Bacillus pumilus MB246, Serratia nematodiphila MB307 and Delftia Lacustris MB322. Pot experiments were conducted with inoculated V. radiata NM-II seeds grown in soil artificially contaminated with lead (Pb), Nickle (Ni) and chromium (Cr) at a concentration of 300, 200 and 100 mg/kg respectively. After harvesting various growth parameters were studied (root length, shoot length, fresh weight and dry weight). Bacterial colonization on root surfaces of harvested plants was observed through Scanning electron microscopy (SEM) and Elemental composition was recorded through Energy dispersive X-ray spectroscopy (EDX) attached with SEM. Metabolic response of harvested plants was studied through Gas chromatography Mass spectrophotometry (GC-MS) analysis. Metal accumulation in roots, shoots and soil was analysed by acid digestion method from which Bioaccumulation factor (BF) and Translocation factor (TF) of metal from soil to plant was calculated. Results revealed stimulatory effect of PGPB on growth and phytoextraction ability of V. radiata. Soil metal removal efficiency was in the order Pb>Ni>Cr, whereas metal distribution in each part of plant was root>stem>leaf. The BF and TF values suggested V. radiata as Pb and Ni excluder while moderate accumulator for Cr. Elemental analysis through Energy Dispersive X- ray spectroscopy (EDX) found potassium (K+)and calcium (Ca+)as highly abundant nutrients with least accumulation of sulphur (S). Metabolites study through GC-MS revealed variety of compounds (carbohydrates, amino acids, fatty acids, steroids etc) detected differentially under each metal treatment and their concentration was influenced by different bacterial inoculations. Overall 9-Octadecenamide was found as commonly present lipid compound in most of the treatments which is required for detoxification in plants. The study concluded beneficial role of PGPB for successful phytoremediation of heavy metals and differential response of metabolites towards each metal stress that is related to metal tolerance ability of V. radiata.

Genetic improvement of peanut (Arachis hypogea L.) genotypes by developing short duration hybrids.

Peanut, the only cash crop of rainfed areas of Pakistan, is facing immense challenges due to global warming. Climatic factors particularly the temperature fluctuations and rain pattern shift significantly impact the production and yield of peanut and unavailability of resilient varieties exacerbate this impact. To deal with the cropping pattern change and yield losses, due to climate vagaries, a study was conducted to develop early maturing hybrids using line into tester mating design. The F1 hybrids from the parental lines were produced in the year 2018 using Line × Tester mating design and then grown in the field in the year 2019 for further evaluation. The hybrids were evaluated based on the early maturity and yield-related attributes in comparison with the parental lines. Based on the general combining ability estimate, line V-3 (Golden), was found as best parent with highly significant values for plant height, days to peg formation, days to maturity, number of pegs per plant, number of pods per plants, number of seeds per plant, 100 pod weight 100 seed weight. Similarly, tester V-7 (PI 635006 01 SD) showed highly significant results of GCA for days to germination, day to 50% flowering, plant height, days to peg formation, days to maturity, number of pegs per plant, number of pods per plants, number of seeds per plant, 100 kernel weight, shelling percentage. All the combinations were evaluated for specific combining ability and significant results were observed for V-3 × V-4 (Golden × PI 619175 01 SD) and V-1 × V-6 (BARI-2000 × PI 564846 01 SD) by developing or maturity and yield-related attributes. The hybrid combinations V-3 × V-5 (Golden × PI 635006 01 SD) followed by V-3 × V-6 showed highly significant results for mid parent heterosis and better parent heterosis for days to 50% flowering, plant height, days to peg formation, number of pegs, days to maturity, number of mature seeds per plant, shelling ratio, 100 pod weight and 100 kernel weight. These parents and hybrid combinations with early maturity genes and high yield attributes can further be used for the development of short duration variety.

Genetic Evidences of Biosurfactant Production in Two Bacillus subtilis Strains MB415 and MB418 Isolated From Oil Contaminated Soil.

Biosurfactants are a diverse group of amphiphilic compounds obtained from microbes. In the present study, the genomic analysis of biosurfactant-producing Bacillus subtilis MB415 and MB418 obtained from oil-contaminated soil was performed. Initially, the strains were screened for biosurfactant production by hemolytic assay, emulsification index, and oil displacement. Further FTIR analysis of extracted biosurfactants revealed the presence of lipopeptides. The sequenced genomes of MB415 and MB418 were of 4.2 Mbps with 43% GC content. Among more than 4,500 protein-coding genes, many were involved in virulence, metal/multidrug resistances, flagella assembly, chemotactic response, and aromatic ring hydroxylating dioxygenases. An annotation analysis revealed that both genomes possessed non-ribosomal synthetase gene clusters for the lipopeptide synthetases srf and fen responsible for surfactin and fengycin production. Comparative studies of both genomes highlighted variability in gene operons mainly for surfactin biosynthesis.

Identification of bacterial communities in extreme sites of Pakistan using high throughput barcoded amplicon sequencing.

Microorganisms thrive nearly everywhere including extreme environments where few other forms of life can exist. Geochemistry of extreme sites plays a major role in shaping these microbial communities and microbes thriving in such harsh conditions are untapped sources of novel biomolecules. To understand the structure and composition of such microbial communities, culture-independent bacterial diversity was characterised for two extreme sites in Pakistan, Khewra salt range and Murtazaabad hot spring. Barcoded amplicon sequencing technique was used to study the microbial communities. Physicochemical analysis of these sites was also conducted to study the dynamics of microbial communities under stressed conditions. Metagenomic sequencing of salt range soil samples yielded of 40,433 16S rRNA sequences, while hot spring sediments produced 76,449 16S rRNA sequence reads. Proteobacteria were predominant in saline soil while Firmicutes were most abundant in hot spring sediment. The taxonomic analysis of saline samples revealed 914 operational taxonomic units (OTUs) while that of hot spring sequences were clustered into 726 distinct OTUs. OTUs from genus Alkalibacillus were most abundant in hot spring sediments, whereas Haloarcula were more prevalent in saline soil. Some unidentified sequences were also present at each taxonomic level. Multivariate analysis indicated that electrical conductivity and pH are the major environmental factors involved in modelling microbial communities. This study revealed a poly-extremophilic microbial community in the Murtazaabad hot spring and characterised the unexplored halophilic microbial diversity of saline soil of Pakistan.

Whole genome sequence analysis reveals high genetic variation of newly isolated Acidithiobacillus ferrooxidans IO-2C.

Acidithiobacillus ferrooxidans, a chemolithoautotrophic bacterium, is well known for its mineral oxidizing properties. The current study combines experimental and whole genome sequencing approaches to investigate an iron oxidizing, extreme acidophilic bacterium, A. ferrooxidans isolate (IO-2C) from an acid seep area near Carlos, TX, USA. Strain IO-2C was capable of oxidizing iron i.e. iron sulphate and iron ammonium sulphate yielding shwertmannite and jarosite minerals. Further, the bacterium's genome was sequenced, assembled and annotated to study its general features, structure and functions. To determine genetic heterogeneity, it was compared with the genomes of other published A. ferrooxidans strains. Pan-genome analysis displayed low gene conservation and significant genetic diversity in A. ferrooxidans species comprising of 6926 protein coding sequences with 23.04% (1596) core genes, 46.13% (3195) unique and 30.82% (2135) accessory genes. Variant analysis showed >75,000 variants, 287 of them with a predicted high impact, in A. ferrooxidans IO-2C genome compared to the reference strain, resulting in abandonment of some important functional key genes. The genome contains numerous functional genes for iron and sulphur metabolism, nitrogen fixation, secondary metabolites, degradation of aromatic compounds, and multidrug and heavy metal resistance. This study demonstrated the bio-oxidation of iron by newly isolated A. ferrooxidans IO-2C under acidic conditions, which was further supported by genomic analysis. Genomic analysis of this strain provided valuable information about the complement of genes responsible for the utilization of iron and tolerance of other metals.

Production, characterization and antimicrobial activities of bio-pigments by Aquisalibacillus elongatus MB592, Salinicoccus sesuvii MB597, and Halomonas aquamarina MB598 isolated from Khewra Salt Range, Pakistan.

Hypersaline ecosystems offer unique habitats to microbial populations capable of withstanding extreme stress conditions and producing novel metabolites of commercial importance. Herein, we have characterized for the first time the production of bioactive pigments from newly isolated halophilic bacterial species. Halophilic bacteria were isolated from Khewra Salt Range of Pakistan. Three distinctly colored isolates were selected for pigment production. Selected colonies were identified as Aquisalibacillus elongatus MB592, Salinicoccus sesuvii MB597, and Halomonas aquamarina MB598 based on morphological, biochemical, and physiological evidences as well as 16S rRNA analysis. The optimum pigment production observed at mesophilic condition, nearly neutral pH, and moderate salinity was validated using response surface methodology. Different analytical techniques (UV spectroscopy, infrared spectroscopy, and HPLC) characterized these purified pigments as derivatives of bacterioruberin carotenoids. Antioxidant activity of pigments revealed up to 85% free-radical scavenging activity at the concentration of 30 µg ml-1. Pigments also showed significant antimicrobial activity against Bacillus subtilis, Bacillus pumilus, Enterococcus faecalis, Bacillus cereus, Klebsiella pneumoniae, Alcaligenes faecalis, Pseudomonas geniculata, Enterococcus faecium, Aspergillus fumigatus, Aspergillus flavus, Fusarium solani, and Mucor spp., suggesting potential biomedical applications.

Production and Biomedical Applications of Probiotic Biosurfactants.

Biosurfactants have been widely used for environmental and industrial applications. However, their use in medical field is still limited. Probiotic biosurfactants possess an immense antimicrobial, anti-adhesive, antitumor, and antibiofilm potential. Moreover, they have an additional advantage over conventional microbial surfactants because probiotics are an integral part of normal human microflora and their biosurfactants are innocuous to human. So, they can be effectively exploited for medicinal use. Present review is aimed to discourse the production and biomedical applications of probiotic biosurfactants.