Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes

Background Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Although vancomycin-resistant Staphylococcus aureus (VRSA) is currently not as widespread as methicillin-resistant S. aureus (MRSA), genome evolution of MRSA into VRSA, including strains engineered within the same patient under anti-staphylococcal therapy, may build up to future public health concern. To further complicate diagnosis, infection control and anti-microbial chemotherapy, non-sterile sites such as the nares and the skin could contain both S. aureus and coagulase-negative staphylococci (CoNS), either of which could harbour mecA the gene driving staphylococcal methicillin-resistance and required for MRSA-VRSA evolution. Results A new heptaplex PCR assay has been developed which simultaneously detects seven markers for: i) eubacteria (16S rRNA), ii) Staphylococcus genus (tuf), iii) Staphylococcus aureus (spa), iv) CoNS (cns), v) Panton-Valentine leukocidin (pvl), vi) methicillin resistance (mecA), and vii) vancomycin resistance (vanA). Following successful validation using 255 reference bacterial strains, applicability to analyse clinical samples was evaluated by direct amplification in spiked blood cultures (n = 89) which returned 100 % specificity, negative and positive predictive values. The new assay has LoD of 1.0x103 CFU/mL for the 16S rRNA marker and 1.0x104 CFU/mL for six other markers and completes cycling in less than one hour. Conclusion The speed, sensitivity (100 %), NPV (100 %) and PPV (100 %) suggest the new heptaplex PCR assay could be easily integrated into a routine diagnostic microbiology workflow. Detection of the cns marker allows for unique identification of CoNS in mono-microbial and in poly-microbial samples containing mixtures of CoNS and S. aureus without recourse to the conventional elimination approach which is ambiguous. In addition to the SA-CoNS differential diagnostic essence of the new assay, inclusion of vanA primers will allow microbiology laboratories to stay ahead of the emerging MRSA-VRSA evolution. To the best of our knowledge, the new heptaplex PCR assay is the most multiplexed among similar PCR-based assays for simultaneous detection of STAAR

ASSESSMENT OF WORKPLACE DIVERSITY ADOPTION IN ACADEMIC STAFF RECRUITMENT BY THE ENUGU STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ENUGU, ENUGU STATE

The focus of the study was on workplace diversity is implemented in the recruitment process for academic staff at Enugu State University of Science and Technology, Enugu. The specific objectives were included to: evaluate the level of ethnic diversity in the recruitment of academic staff, examine the degree of gender diversity in the recruitment of academic staff and assess the extent of religious diversity in the recruitment of academic staff. A survey approach was adopted. Sample size was determined using Freund and Williams's formula, resulting in a sample of 462 out of a population of 712. Out of the distributed questionnaires, 366 staff members accurately completed and returned them, resulting in a response rate of 79 percent. Content analysis was employed to assess the validity of the instrument, yielding satisfactory results. The reliability of the instrument was assessed using the Pearson correlation coefficient (r), which indicated a good reliability coefficient of 0.76. For hypothesis testing, the data were analyzed using the Z-test, with the assistance of statistical software, specifically the Special Package for Statistical Software (SPSS). The findings revealed that the adoption of ethnic diversity in the recruitment of academic staff at Enugu State University of Science and Technology is not significantly low (Z(95, n=366) = 4.011 < 5.101, p<.05), gender diversity is not significantly incorporated in the recruitment of academic staff at Enugu State University of Science and Technology, Enugu (Z(95, n=366) = 2.610 < 3.564, p<.05) and the recruitment of academic staff at Enugu State University of Science and Technology demonstrates significant adoption of religious diversity (Z(95, n=366) = 4.353 < 5.233, p<.05). We concluded that workplace diversity, particularly in terms of ethnicity, gender, and religion, significantly influences the recruitment of academic staff at Enugu State University of Science and Technology, Enugu. Embracing a diverse workforce enhances innovation, creativity, and overall performance. The study recommended that Enugu State University of Science and Technology (ESUT) should implement flexible and appropriate strategic planning practices to foster ethnic diversity in the recruitment of academic staff, which will contribute to enhancing the overall performance of the universit

Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for Diagnostic and Therapeutic Applications

Abstract Background: Staphylococcus aureus produces several toxins, including Panton-Valentine leukocidin (PVL). The involvement of PVL in primary skin infections, necrotizing pneumonia, musculoskeletal disorders, brain abscess, and other diseases, some of which are life-threatening, has been reported. Following expert opinion, we aimed to provide the tools for establishment of sequence-based diagnostics and therapeutics for those conditions. We engineered the synergistic S and F (LukS-PV and LukF-PV respectively) pro-toxin subunits from Staphylococcus aureus USA400 into separate expression E. coli BL21(DE3)-pLysS hosts. Results: Following Nickel affinity chromatography (NAC), the F subunit came out without bands of impurity. The S sub-unit did not come off very pure after NAC thus necessitating further purification by size exclusion and ion-exchange chromatography. The purification plots showed that the BioLogic-LP and AKTA systems are reliable for following the progress of the chromatographic purification in real-time. Computer predicted Mw for the 6His-LukF-PV and 6His-LukS-PV were 35645.41 Da and 33530.04 Da respectively, while the mass spectrometry results were 35643.57 Da and 33528.34 Da respectively. Conclusion: The BioLogic-LP and AKTA systems are commendable for reliability and user-friendliness. As a recent work elsewhere also reported that a second round of chromatography was necessary to purify the S subunit after the first attempt, we speculate that the S subunit might contain yet unidentified motif(s) requiring further treatment. The purified S and F sub-units of PVL were supplied to the Nottingham Cancer Immunotherapy group who used them to establish sequence-based monoclonal antibodies for diagnostic and therapeutic uses targeting PVL