The Use Of Rap-PCR In Studying Mycobacterium tuberculosis Intracellular Gene During Macrophage Infection

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes, which were specifically expressed, or upregulated during intracellular infection ofJ774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine macrophage cell line was infected with M. tuberculosis (H37Rv strain) at 10:1 multiplicity of infection (MOI). RNA wasdifferentially extracted from M. tuberculosis infecting J774 macrophage cell line. The control in this case was RNA from extracellular broth grown bacteria. Approximately 50 ng of RNA from intracellular derived bacteria and extracellular derived bacteria (control) were subjected to random arbitrarily primed PCR (RAP-PCR) using 50 primer combinations. Eleven differential RAP-PCR products were observed. All RAP-PCR products were cloned into pCR®2.1 and sequenced in order to determine the identity of the products. Four of the eleven products were derived from macrophage genes and another 4 products were derived from the M. tuberculosis rRNA genes (three 23S and one 16S rRNA). The 3 remaining RAP-PCR products were found to be mycobacterial genes other than ribosomal genes. The three products were genes encoding enzyme involving in a shikimate pathway, a putative carboxyphosphonoenolpyruvate phosphonomutase and a serine protease with homology to HtrA. Of the 3 mycobacterial genes other than ribosomal genes detected, none were specifically expressed during intracellular infection but competitive RT-PCR showed that aroF gene was upregulated two-fold in intracellular derived bacilli

In-Vitro efficacy of antimicrobial agents used in the treatment of bacterial eye infections in Ibadan, Nigeria

Failure to cure eye infections, and reduced potency in topical antimicrobials had been observed in South Western Nigeria, this study sought to evaluate in vitro, the efficacy of antimicrobial agents in the treatment of ocular infections. A total of 46 ocular bacterial isolates were recovered from the diagnostic laboratory of the University College Hospital, Ibadan, from conjunctival swabs of patients having underlying eye diseases (Cataracts, glaucoma and esotrapia), and from patients presenting with other symptoms of eye infections. The pathogens incriminated were Staphylococcus aureus (73.5%), Coagulase negative Staphylococci (13.3%), Klebsiella species (10.3%), and Pseudomonas aeruginosa (2.0%). Disc diffusion tests (Bauer-Kirby method) were carried out using ciprofloxacin, gentamicin, chloramphenicol, erythromycin, augmentin, cefuroxime and levofloxacin. Broth dilution techniques were thereafter performed using gentamicin, chloramphenicol and ciprofloxacin. The microlide- erythromycin was 63.0% efficacious, augmentin and cefuroxime showed 71.1% and 76% efficacy. Minimum inhibitory concentrations (MIC) of commonly used topical antibiotics however showed different levels of resistance. Resistance to the aminoglycosides was marked, yielding 53.4% with MIC50= 8, MIC90 > 256, Resistance to chloramphenicol was even more marked 69.6% with MIC50= 16, MIC90= 64, the fluoroquinolones showed high efficacy- levofloxacin and ciprofloxacin showed 93.4% and 82.6% susceptibility respectively with MIC50 < 0.5, though slightly demonstrable resistance was observed (MIC90= 8). The study thus recommends the discontinuation of empirical therapy by physicians in order to stem the tide of resistance; it justifies the inclusion of the fluoroquinolones in susceptibility testing of ocular bacterial isolates, and its first line of choice if cure is warranted.doi: 10.4314/ajcem.v12i3.

In vitro Evaluation of antibacterial agents against ocular Bacterial isolates from a Tertiary Hospital, South-West of Nigeria

ABSTRACT Failure to cure eye infections and reduced potency in ocular antibacterial agents had been observed in South Western Nigeria, this study sought to evaluate in vitro, the efficacy of antibacterial agents used in the treatment of eye infections. A total of 135 bacterial isolates were recovered from the diagnostic laboratory of the University College Hospital, Ibadan, from conjunctival swabs of patients having underlying eye diseases (Cataracts and glaucoma), and from patients presenting with other symptoms of eye infections (conjunctivitis, keratitis and dacryocystitis). The pathogens incriminated were Staphylococcus aureus (75.5%), Coagulase negative Staphylococci (11.1%), Klebsiella species (11.1%), and Pseudomonas aeruginosa (2.2). Disc diffusion tests (Kirby-Bauer method) were carried out using ciprofloxacin, gentamicin, chloramphenicol, erythromycin, amoxicillinclavulanate, cefuroxime and levofloxacin. Broth dilution technique was thereafter demonstrated using gentamicin, chloramphenicol and ciprofloxacin. The macrolideerythromycin was 54.4% efficacious, amoxicillin-clavulanate and cefuroxime showed 69.9% and 72.8% efficacy. Minimum inhibitory concentrations (MIC) of commonly used antibiotics however showed different levels of resistance. Resistance to the aminoglycosides was marked, yielding 51.6%, with MIC 50 = 8, MIC 90 &gt; 256, resistance to chloramphenicol was also marked, yielding 76.9%, with MIC 50 = 8, MIC 90 = 64. The fluoroquinolones showed high efficacy; levofloxacin and ciprofloxacin showed 91.1% and 75.5% susceptibility respectively, with MIC 50 &lt; 0.5, though slightly demonstrable resistance was observed (MIC 90 = 8). This study recommends discontinuation of empirical therapy by physicians in order to stem the tide of resistance; it justifies the inclusion of the fluoroquinolones in susceptibility testing of bacterial isolates and its first line of choice if cure is warranted

Co-agulase Negative Staphylococcus Distribution in Clinical Sample in a Tertiary Hospital in Ibadan, Nigeria

Numerous researchers have described the isolation of coagulase-negative staphylococci especially S. epidermidis, and the association of the bacteria with clinical disease. In this study we determined the distribution of coagulase-negative staphylococci isolated from clinical samples in tertiary hospital. One hundred and fifteen repeat CoNS isolates were obtained from 607 various clinical specimens using standard precedures; 97 (84.3%) were strains of S. epidermidis while 18 (15.7%) were S. Saprophyticus. The highest number of isolates of CoNS were from Blood culture, 75 out of 115 strains (65.2%) and 68.0% were strains of S. epidermidis. Septicaemia had the highest prevalence of CoNS, 31.4%; followed by endocraditis, 25.0%. The least was found in otits and pyrexia of unkonwn origin, 7.4% and 8.3% respectively. All the CoNS strains were resistant to one or more of the eleven antimicrobial agents used. The frequancy of susceptibility to fluoroquinolones was the highest; ofloxacin(81.7%), ciprofloxacin (77.4%). The least susceptibility was found in tetracycline; 20.0%. No isolate was sensitive to cotrimoxazole. However, S. epidermidis had a greater percentage of strains susceptibleto the quinolones; ofloxacin; 84.0% and ciprofloxacin; 80.0% than the S. saprohyticus, ofloxacin; 67.0% and ciprofloxacin; 44.0%. The distribution of CoNS in clinical specimens obtained from hospital enivronment in this study has shown that CoNS have become an important pathogen, therefore antimicrobial susceptibility tests should be carried out as a guide for therapy and to control development of resistant strains