Biocontrol of MRSA and E. coli using bacteriophages from cow manure

Background: Bacteriophages can be used as biocontrol agents to eliminate the undesirable and targeted bacteria in human beings, environment and industries. We aimed to isolate phages from cow manure and investigate their antibacterial and anti-biofilm formation effect against MRSA and E. coli species.Method: E. coli and S. aureus isolates of clinical origin were sub-cultured on MacConkey’s and Mannitol salt agar media, respectively. To confirm the Methicillin resistance in S. aureus, mecA gene was detected by the PCR using gene specific forward and reverse primers. The bacteriophages were isolated using enrichment procedure from cow manure. Their antibacterial and anti-biofilm formation activity was determined through the inhibition of bacterial growth and decrease in the absorbance of Crystal Violet through biofilm inhibition assay, respectively.Results: The isolated phages showed clear plaque formation against MRSA and E. coli species. The time-dependent lytic assay showed a sharp decline in OD600 of MRSA and E. coli after one hour incubation with the phages. Biofilm inhibition was indicated by a decrease in the absorbance (OD595) of CV in a microtiter well plateConclusion: This is perhaps the first study to isolate phages from cow manure at local region and demonstrate their lytic efficiency against MRSA and E. coli, with the ultimate aim to use them as a promising biocontrol agent against antibiotic resistant pathogens.Keywords: Phages; MRSA; Plaque formation; Biofilm inhibition; E. coli; Biocontro

A cost-effective o-toulidine-based Schiff base as an efficient sorbent for metal ion uptake from aqueous and soil samples: Synthesis, antimicrobial, and acute toxicity analyses

Heavy metals create serious health problems, so the practical implementation and development of low-cost sorbent materials to remove heavy metals from the ecosystem is a worldwide issue. The purpose of this study is to find a low-cost ligand that has the potential to adsorb heavy metals from aqueous and soil samples and also has biological potential. For this, a Schiff base, dimeric o-toluidine (SBL), has been synthesized through condensation, characterized by spectroscopic analysis, and had its biological activities measured. We also studied its adsorption efficiency through a batch technique to remove Zn(II), Co(II), and Cu(II) from aqueous and soil samples under different conditions such as metal ion concentration, pH, contact time, and SBL concentration. The adsorption potential of SBL was analyzed by the Langmuir and Freundlich adsorption isotherms. The values of correlation coefficients revealed that the Freundlich isotherm elucidated results that were more appropriable than the Langmuir model. Adsorption equilibrium was established in 90 min for aqueous samples and in 1,440 min for soil samples. For the maximum adsorption of all metals, the optimum pH was 8, and it showed a capacity to remove 77 to 95 percent of metals from the samples. The maximum adsorption capacity (qmax) of SBL were 75.75, 62.50, and 9.17 mg g-1 in the case of Cu(II), Zn(II), and Co(II) ions, respectively, from aqueous samples and 10.95, 64.10, and 88.49 mg g-1 in the case of Zn(II), Cu (II), and Co(II), respectively, from soil samples. The effectiveness of SBL in the sorption of the selected metals was found to be Cu+2 > Zn+2 > Co+2 for aqueous samples and Co+2 > Cu+2 > Zn+2 for soil samples. The antimicrobial activity of SBL was also investigated. The results revealed that SBL showed moderate inhibitory activity against Staphylococcus dysentria, C. albican, and Aspergillus niger, whereas it exhibited weak activity against S. aureus, P. aureginosa, K. pneumoniae, P. vulgaris, and E.coli when compared to Fluconazole and Ciprofloxacin as the standard. Acute toxicity of the synthesized compound was measured through its daily oral administration with various doses ranging from 0.1 to 1,000 mg/kg of the mice’s body weights. Even at the dose of 1,000 mg/kg, the SBL showed no mortality or any type of general behavioral change in the treated mice. Based on preparation cost, metal removal capacity, toxicity, and antimicrobial activities, SBL is an excellent sorbent and should be studied at pilot scale levels

Complete genome sequencing and analysis of Pasteurella multocida strain PMTB2.1 and expression of selected genes in iron-restricted environment

Pasteurella multocida (PM) is a Gram-negative, facultative anaerobic bacterium, belonging to the family Pasteurellaceae that commonly found as commensal in the upper respiratory tract of mammals and birds. However, P. multocida is often associated with acute as well as chronic infections in avian and bovine leading to significant morbidity and mortality, such as pasteurellosis and hemorrhagic septicemia (HS) in cattle and buffaloes. P.multocida subspecies multocida strain PMTB2.1 was first isolated from buffalos died of septicemia. The bacterium has been characterized based on biochemical tests and molecular identification based onPCR. Intrestingly, based on HS causing serogroup B-specific PCR (HSB-PCR), the isolate is not from serogroup B. Hence, an in depth genome wide analysis of PMTB2.1 was carried out. In this study, the genome of P. multocida strain PMTB2.1 was sequenced using third-generation sequencing technology, PacBio and analysed bioinformatically via de novo method followed by in depth characterization of the genome. In addition, expression of selected genes of PMTB2.1 grown in iron-restricted condition was also demonstrated based on real-time PCR study. Bioinformatics analysis based on de novo assembly of PacBio raw reads generated 3 supercontigs that were assembled to generate a draft genome with unresolved gaps regions. The gaps between the contigs in the assembled draft genome sequence were closed by PCR sequencing with primer walking strategy using Sanger sequencing. Start position of the circular genome of PMTB2.1 was set based on homology to reference genome P. multocida strain PM36950 and the circularity of the genome was confirmed by PCR. The complete genome sequence of P. multocida strain PMTB2.1 is composed of a single circular chromosome of 2,315,138 base pairs with 40.32 % GC content and a total of 2,176 potential genes. The genome was submitted to NCBI under the accession number, CP 007205.1. The annotated complete genome sequences of PMTB2.1 have 2,097 protein-coding sequences, 19 rRNA genes, 56 tRNA and 4 ncRNA genes. The genome also encode for more than 41 CDS (2%) that involved in iron regulation or iron uptake, 160 virulence genes and 12 antibiotic resistance genes including the complete Tad locus. The tad locus encodes 14 gene including several previously uncharacterized genes such as flp 2 that play important roles in the adhesion and colonization of the bacteria, biofilm formation as well as in pathogenesis of the disease. Multi-locus sequence typing against Rural Industries Research and Development Corporation (RIRDC) scheme indicated that PMTB2.1 matched to alleles from sequence type ST101. Comparative genome analysis showed that PMTB2.1 is closely related with other Pasteurella multocida strains with genomic distance less than 0.13. However, synteny analysis showed that genome structure of PMTB2.1 is more resembles to that of P. multocida serogroup A strain PM36950 as compared to that of P. multocida serogroup F strain PM70. However, PMTB2.1 genome lacks the Integrative Conjugative Element (ICE) of 86 kb that can only be detected in PM36950. Nevertheless, two intact prophage sequences of approximately 62 kb that were found in PMTB2.1, were absent in PM36950 and PM70. One of the phages is similar to transposable Mu like phage SfMu; however, the phage regions of PMTB2.1 were not associated with toxin-related genes, as detected in serogroup D toxigenic strain of P. multocida. Moreover, PMTB2.1 complete genome is approximately 34,380 kb smaller than PM36950 genome (2,349,518 bp), on the other hand approximately 15 kb specific region of PMTB2.1was absent in PM70 genome. The capsular sequence analysis of PMTB2.1 indicated that it is resembling the capsular sequence of P. multocida serogroup A with 99% sequence identity with A:1 capsular sequences. Furthermore, OrthoMCL analysis based on similarity among common genes showed that PMTB2.1 was clustered with bovine isolates and were separated from other P. multocida strains that infect avian and swine. Since P. multocida including PMTB2.1 has more than 2% of the genome encode for iron-regulated genes, the expression profiling of iron uptake genes namely fbpb, yfea, fece, fur and sialidase encoded by nana were characterized under iron-restricted environment where PMTB2.1 was grown in broth with and without iron chelating agent 2,2’ Bipyridine. Results of this study reflect that iron-reduced conditions have significant effect on the expression profiles of iron-regulating genes (p<0.05) and all of the four iron-related genes (fbpb, yfea, fece, fur) behave differently in response to iron reduction in media. The highest relative fold change (281.2 fold) of fece gene was observed at early, 30 minutes of treatment reveal that P. multocida may utilizes its periplasmic protein at early stage to acquire iron. Furthermore, down-regulation expression of fece from 4 to -1.5 with the elevated expression of other genes (fbpb and yfea) at later time points, 60 and 120 minutes suggest that PMTB2.1 control their iron requirements in response to iron availability by down regulating the expression of iron proteins. Moreover, the significant increase (p≤ 0.05) in fbpb expression (25 fold) at time point 60 and in Yfea expression (26 fold) at early time point 30 minutes with highest expression (42 fold) at 120 minutes reflect the utilization of multiple iron systems in P. multocida strain PMTB2.1. These results demonstrate the importance of iron in the survival of P. multocida. In conclusion, this study has provided insight on the genomic structure of PMTB2.1 in terms of potential genes that can functions as virulence factors and comparative pathogenomic information of valuable importance for future study in elucidating the mechanisms behind the ability of the bacterium in causing diseases in susceptible animals

Analysis of Physical, Chemical and Biological Aspects of Drinking Water at University of Sindh Jamshoro: Short Communication

Safe drinking water is a basic need for good health. It is critically needed to characterize drinking water, by measuring its pH and electrical conductivity, total dissolved solids, and analysing for biological contamination. Drinking water quality standards are set with some parameters and that harmful constituents should not exveed (WHO guidelines (2011)

Hazard and operability study of condensate oil refinery

Petroleum refineries are multifaceted and complex systems that perform multiple phase operations characterized by a high level of risk. The size and complexity of petroleum refinery, together with the nature of the products handled, require Hazard and operability (HAZOP) methodology. HAZOP is one of the best tools used in various oil refineries globally based on guidewords for all hazardous conditions with strong recommendation. The application of HAZOP has been demonstrated in the condensate oil exploration unit, based in Sindh. In this research paper about 12 deviations were documented with 34 possible causes and 46 predicted consequences. However, to make the system hazard free 3 nodes were selected on the piping and instrumentation diagram of condensate splitter, based on selected nodes 48 actions were proposed in counter of the deviations

The chloroplast genome of Chrozophora sabulosa Kar. & Kir. and its exploration in the evolutionary position uncertainty of genus Chrozophora

Abstract Chrozophora sabulosa Kar. & Kir. is a biennial herbaceous plant that belongs to the Euphorbiaceae family and has medicinal properties. This research aimed to identify the genetic characteristics and phylogenetic position of the Chrozophora genus within the Euphorbiaceae family. The evolutionary position of the Chrozophora genus was previously unknown due to insufficient research. Therefore, to determine the evolutionary link between C. sabulosa and other related species, we conducted a study using the NGS Illumina platform to sequence the C. sabulosa chloroplast (cp.) genome. The study results showed that the genome was 156,488 bp in length. It had a quadripartite structure consisting of two inverted repeats (IRb and IRa) of 24,649-bp, separated by an 87,696-bp LSC region and a 19,494-bp SSC region. The CP genome contained 113 unique genes, including four rRNA genes, 30 tRNA genes, and 79 CDS genes. In the second copy of the inverted repeat, there were 18 duplicated genes. The C. sabulosa lacks the petD, petB, rpl2, and rps16 intron. The analysis of simple sequence repeats (SSRs) revealed 93 SSR loci of 22 types and 78 oligonucleotide repeats of four kinds. The phylogenetic investigation showed that the Chrozophora genus evolved paraphyletically from other members of the Euphorbiaceae family. To support the phylogenetic findings, we selected species from the Euphorbiaceae and Phyllanthaceae families to compare with C. sabulosa for Ks and Ka substitution rates, InDels investigation, IR contraction and expansion, and SNPs analysis. The results of these comparative studies align with the phylogenetic findings. We identified six highly polymorphic regions shared by both families, which could be used as molecular identifiers for the Chrozophora genus (rpl33-rps18, rps18-rpl20, rps15-ycf1, ndhG-ndhI, psaI-ycf4, petA-psbJ). The cp. genome sequence of C. sabulosa reveals the evolution of plastid sequences in Chrozophora species. This is the first time the cp. genome of a Chrozophora genus has been sequenced, serving as a foundation for future sequencing of other species within the Chrozophoreae tribe and facilitating in-depth taxonomic research. The results of this research will also aid in identifying new Chrozophora species