Charles Okolie

Helicobacter pylori mutants defective in the clpP ATP-dependant protease and the chaperone clpA display reduced macrophage and murine survival.

The ATP-dependent caseinolytic proteases (Clp) are important in resistance against environmental stresses, antibiotic treatments and host immune defences for a number of pathogenic bacteria. ClpP is the proteolytic subunit, whilst ClpA acts both as a chaperone and as an ATPase driving the degradation of damaged or mis-made proteins. The gastric pathogen Helicobacter pylori infects approximately half of the worlds population and can cause gastric or duodenal ulcers, gastric malignancies and mucosa-associated lymphoid tissue lymphomas. The conditions of its in vivo environment expose the organism to host immune cells and upon treatment, antibiotics, conditions likely to cause protein damage. We generated isogenic nonpolar mutants in strain SS1 of clpP and clpA and double mutants with both genes inactivated. Such mutants showed increased sensitivity to antibacterials causing protein damage and/or oxidative stress, in addition to a reduced survival in human macrophages. In the mouse infection model the double mutant SS1 clpAP lacked all ability to colonize the murine host. This suggests that the ability to recover from protein damage is of key importance in the pathogenesis of this organism.

Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers

Staphylococcus aureus strains harbouring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbour mecA, the gene encoding staphylococcal methicillin-resistance. There have been previous attempts at distinguishing MRSA from MRCoNS, most of which were based on the detection of one of the pathognomonic markers of S. aureus, such as coa, nuc or spa. That approach might suffice for discrete colonies and mono-microbial samples; it is inadequate for identification of clinical specimens containing mixtures of S. aureus and CoNS. In the present study, a real-time pentaplex PCR assay has been developed which simultaneously detects markers for bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl) and methicillin resistance (mecA). Staphylococcal and non-staphylococcal bacterial strains (n = 283) were used to validate the new assay. The applicability of this test to clinical samples was evaluated using spiked blood cultures (n = 43) containing S. aureus and CoNS in mono-microbial and poly-microbial models, which showed that the 5 markers were all detected as expected. Cycling completes within 1 h, delivering 100% specificity, NPV and PPV with a detection limit of 1.0 × 10(1) to 3.0 × 10(1) colony forming units (CFU)/ml, suggesting direct applicability in routine diagnostic microbiology. This is the most multiplexed real-time PCR-based PVL-MRSA assay and the first detection of a unique marker for CoNS without recourse to the conventional elimination approach. There was no evidence that this new assay produced invalid/indeterminate test results.

Hemoglobin and Serum Iron Concentrations in Menstruating Nulliparous Women in Jos, Nigeria

Background: Low hemoglobin (Hb) and iron deficiency among child bearing females have been linked to decreased immune system function, impaired cognitive functioning and complications in pregnancy. Methods: A total of 106 blood samples from apparently healthy nulliparous female students were assayed for Hb and serum iron concentrations using the cyanmethemoglobin and bathophenanthroline methods, respectively, to evaluate changes that may occur in these parameters at different phases of the reproductive cycle. Results: The mean (SD) Hb values during the ovulatory, menstrual, and follicular phases were 13.27 (1.14) g/dL, 12.05 (1.31) g/dL, and 12.23 (1. 56) g/dL, respectively. The prevalence of anemia (Hb<12 g/dL) was reported among 21 (19.8%) subjects, and 31 subjects declined to complete their samples collection. The mean serum iron concentrations during the 3 phases were 92.98 (18.25) µg/dL, 79.90 (13.14) µg/dL and 70.85 (18.65) µg/dL, respectively. A total of 28 (26.4%) study participants showed iron deficiency (serum level, <65 µg/dL). These variations in the values of Hb and serum iron concentrations were statistically significant in the 3 phases. However, no significant difference was observed in Hb concentrations between the menstrual and follicular phases. Of interest, a positive correlation was observed between the hemoglobin and serum iron concentrations within the phases, with the exception of a few cases that showed negative correlations. Conclusion: Menstruation has been shown to be the major cause of anemia and iron deficiency in nulliparous women. A prophylactic dose of iron and folate supplements may be indicated for menstruating females to cushion the adverse effects of menstruation on hematologic status.

Gene expression of Aurora kinases in prostate cancer and nodular hyperplasia tissues.

Objective: To measure the transcript levels of Aurora kinases and compare them to their immunoreactivity patterns in prostate tumors. Materials and Methods: A total of 26 cases of prostate cancer (PCa) and 38 cases of benign nodular hyperplasia (BPH) were sampled from archived formalin-fixed paraffin-embedded tissues. Tissue sections were lysed, total RNA extracted and cDNA made by random hexamer priming while slide sections were immunostained for the kinases. Normalized relative quantitation was performed for all the kinases using real-time PCR on TaqMan chemistry. Results: The immunoreactivity profile showed 15.4, 53.8 and 30.7% positivity for Aurora kinases A, B and C in PCa cases, respectively, while the positivity was 76.3, 73.7 and 84.2% for the same kinases in BPH cases. The immunoreactivity was preponderant on epithelial tissue compared to stromal component. Conclusion: Aurora kinases were significantly overexpressed in BPH cases compared to PCa cases. At the transcript level, there was no significant differential expression in the kinases between PCa and BPH cases.

Optimization and Evaluation of Triplex Real-time PCR Assay for Detection of Genes Encoding Staphylococcal Virulence and Methicillin Resistance Using Two Different Multi-channel Emission Instruments

Aim of Study: To optimize a triplex real-time PCR assay developed elsewhere and to evaluate the performance characteristics of two different multi-channel real-time PCR systems on the newly optimized assay. Methodology: A triplex real-time PCR assay developed for three key genes encoding virulence and antibiotic resistance in Staphylococcus aureus, namely, lukSF-PV, mecA, and spa was optimized and evaluated using two different real-time PCR instruments (7500SDS and LightCycler 480). Bacterial strains (N=230), including staphylococcal and non-staphylococcal isolates, were Original Research Article Okolie and James; JALSI, 2(4): 145-151, 2015; Article no.JALSI.2015.016 146 used for the study. Results: Following optimization, cycling and data analysis completed within one hour compared with the former three hours. Assay specificity became 100% on both instruments. The negative predictive value (NPV) and the positive predictive value (PPV) rose to 100%. Conclusion: The optimized triplex real-time PCR assay is highly reproducible across the two systems without loss of speed, sensitivity and specificity. The results also suggested that userfamiliarization with each machine operational system would allow assay performance across various real-time PCR platforms currently being used.

Development of New Pentaplex PCR Assay for Differentiating Staphylococci from Other Bacteria with Simultaneous Detection of Staphylococcus aureus Genes Encoding Panton-Valentine Leukocidin and Methicillin Resistance

In some human infections including those of blood, skin and respiratory tract, the causative bacterial agents tend to overlap, especially Staphylococcus spp. and Streptococcus spp. Such overlaps constitute difficulties in the choice of diagnostic tools, antibacterial chemotherapy and infection control strategies. To resolve this challenge, we developed a pentaplex PCR assay which simultaneously detects sequences for the recognition of bacteria (bacterial 16S rRNA), the genus staphylococcus translation elongation factor Tu (tuf), Staphylococcus aureus (spa), mecA-encoded Original Research Article Okolie and James; JABB, 2(4): 250-259, 2015; Article no.JABB.2015.025 251 staphylococcal methicillin resistance (mecA) and the S. aureus Panton-Valentine leukocidin (PVL) virulence factor (pvl). The new pentaplex PCR assay was validated using standard bacterial strains (N=377) including strains from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA), the National Collection of Type Cultures (NCTC), and the National Collection of Industrial and Marine Bacteria (NCIMB). The new pentaplex PCR assay enables inference of bacterial presence/absence, differentiates between the genus Staphylococcus spp. and other bacteria, separates S. aureus from other Staphylococcus spp., differentiates between methicillin-susceptible and methicillin-resistant staphylococci, and detects the S. aureus PVL gene locus. The negative predictive value was 100% while the positive predictive value was 100%. Using a 96-well plate, the time to result was 2.5 hours against ≥24 hours by bacteriological culture. The new pentaplex PCR assay can easily be integrated into routine diagnostic microbiology workflow especially for laboratories with slim budgets which are unable to incorporate next generation sequencing at the moment.

Genetic and Phenotypic Identification of Vancomycin-Resistant Staphylococcus aureus Isolates from Retail Poultry Carcasses in Omu-Aran, North-Central Nigeria.

Staphylococcus aureus is a well-known agent of zoonotic infections. Livestock-associated methicillin-resistant S. aureus (LA-MRSA) had been receiving public health attention for over a decade. Recently, the genomes of some MRSA strains evolved further by enabling acquisition of vanA gene from enterococcus which drives the emergence of vancomycin-resistant S. aureus (VRSA), thus signaling a higher threat to antimicrobial chemotherapy and diagnostic microbiology. This study was designed to examine slaughtered chicken carcasses in Omu-Aran, North-Central Nigeria for the presence of VRSA using vancomycin agar screen (VAS) as recommended by the Clinical and Laboratories Standards Institute (CLSI). To provide independent witness to further support the evidences from VAS, a 235 bp marker for vanA gene was simultaneously detected by Original Research Article Okolie et al.; BBJ, 6(2): 87-92, 2015; Article no.BBJ.2015.030 88 PCR. From April 2013 through May 2014, chicken carcasses (n=784) were collected and studied. Among 155 (19.8%) samples which yielded S. aureus, VAS and vanA PCR methods unequivocally identified VRSA in 22 (14.2%). Compared with 46.2% VRSA report from Zaria, North-Western Nigeria, the incidence of VRSA is much less in Omu-Aran chicken carcasses than those of Zaria. Further investigation in other parts of Nigeria is recommended in order to generate nation-wide data on VRSA in this country.