Materials used

Test tubes, test tube rack, conical flasks, sterile syringes, pipettes, Durham tubes, McCartney bottles, wire loops, Petri dishes, beaker, autoclave, incubator, oven, microscope slide, weighing balance, cotton wool, measuring cylinder, Bunsen burner, refrigerator and spectrophotometer were used.

Sterilization of materials

All glass wares used for this research were thoroughly washed with detergent and rinsed under clean running water. Thereafter, glass wares were sterilized in the hot air oven at 180 °C for an hour, and the wire loop was flamed to redness before and after use.

Collection and identification of etiology of bacteremia

The etiology of bacteremia in HIV-infected patients was identified by the Vitek 2 automated system (Biomerieux Inc., France) and as well by conventional biochemical tests. The results obtained were compared with databases for bacterial isolates in Bergey’s Manual of Systematic Bacteriology, were used in this study (Holts et al. 1994). The isolates comprised of Staphylococcus aureus (n = 2), Streptococcus pneumoniae (n = 2), Acinetobacter spp (n = 1), Salmonella typhi (n = 2), Klebsiella pneumoniae (n = 2), Enterobacter aerogenes (n = 1), Haemophilus influenzae (n = 1), and Pseudomonas aeruginosa (n = 1). These isolates were obtained from the Microbiology Laboratory, University of Uyo, Akwa Ibom State.

Source of fluoroquinolone antibiotics

Ofloxacin (OFL 500 mg, Ronald Pharmaceuticals Pvt, Vadodara, India); Levofloxacin (LEV 500 mg, Zee Laboratory, India); and Ciprofloxacin (CIP 400 mg, Jiangsu Ruinian Pharmaceuticals Ltd, China) were purchased in tablet form from standard pharmacy stores in Uyo. Stock solutions (10 mg/mL) of OFL, LEV and CIP were prepared using sterile distilled water (dH20) as the solvent and stored at 4 °C prior to each experiment.

Antibacterial activities of fluoroquinolones against etiology of bacteremia

The antibacterial activities of fluoroquinolone antibiotics: OFL, LEV and CIP against etiology of bacteremia in HIV-infected patients were determined by disc diffusion method (CLSI 2018; Akinjogunla et al. 2021). The isolates used were S. aureus (SA08, SA21); S. pneumoniae, (SP02, SP10); Acinetobacter spp (AS01); S. typhi (ST07, ST40); K. pneumoniae (KP26, KP32); E. aerogenes (EA01); H. influenzae (HI27) and P. aeruginosa (PA09). Mueller–Hinton agar (MHA) plates were aseptically prepared and 100 µL of each bacterial inoculum, prepared directly from an overnight nutrient agar plate and adjusted to 0.5 McFarland Turbidity Standard (corresponding to approximately 106 CFU/mL), was inoculated onto each MHA plate and thereafter evenly spread using a sterile spreader. Each test antibiotic (OFL, LEV and CIP) was dissolved in dH20 to achieve graded concentrations of 2.5 and 5 mg/mL. Each sterile filter paper disc of 6 mm diameter was impregnated with 10 μL of 2.5 and 5 mg/mL test antibiotic. The impregnated discs were carefully placed on to MHA plates which had previously been inoculated with the isolates and were incubated at 37 °C for 24 h. A disc containing 10 μL of dH20 that served as a solvent control was included in each plate. The same procedure described above was repeated for LEV and CIP. The experiments were performed in independent triplicates to validate the results, and the mean zones of inhibition diameter in millimeters were determined.

Evaluation of minimum inhibitory and minimum bacteriocidal concentrations of fluoroquinolones

The minimum inhibitory concentration (MIC) values of fluoroquinolone antibiotics: OFL, LEV and CIP against etiology of bacteremia in HIV-infected patients were determined using micro-broth dilution technique (CLSI 2018). Five hundred (500) mg of OFL, LEV and CIP were separately dissolved into 50 mL of dH20 to give a concentration of 10 mg/mL. One milliliter (mL) of the stock solution (10 mg/mL of OFL, LEV and CIP) was serially diluted in sterile dH20 by twofold dilution to achieve the range of test concentrations of 5—0.625 mg/mL for each antibiotic solution. To 100 µL of varying concentrations of OFL (0.625, 1.25, 2.5, 5 and 10 mg/mL) in test tubes was added nutrient broth (9.9 ml) to give the final concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL for the MIC testing and a loopful of each prepared bacterial isolate was added. A tube comprising dH20 with inoculum bacterial cells served as control. The same procedure described above was repeated for LEV and CIP. All the culture tubes were incubated at 37 °C for 24 h and thereafter the tubes were examined for microbial growth (turbidity measured using spectrophotometer). The MIC was taken as the lowest concentration of OFL, LEV and CIP that visibly inhibited the bacterial growth after 24 h of incubation.

For the minimum bacteriocidal concentration (MBC), the aliquot (1 mL) from each of MIC broth tubes without visible growth was inoculated onto each of the sterile nutrient agar plates using sterile pipette and streaked. The inoculated plates were inverted and incubated at 37 °C for 24 h. After incubation, the least concentration of the OFL that killed the bacterial isolate was observed and considered as the MBC value. The same procedure was repeated for LEV and CIP as described above.

Time-kill bactericidal evaluation of fluoroquinolones against etiology of bacteremia

The time-kill evaluation of fluoroquinolone antibiotics: OFL, LEV and CIP against etiology of bacteremia was carried out using macro broth dilution and pour plate techniques (CLSI 2018; Agbo et al. 2020). An overnight nutrient broth culture of each isolate, adjusted to 0.5 McFarland turbidity standard to obtain a starting inoculum between 105 and 106 CFU/mL (confirmed by quantitative plate counts), was used. The tubes containing the isolates were shaken at 150 rpm for 90 min at 37 °C to ensure that isolates were in their early exponential phase of growth. One (1) millilitre of each exponentially growing isolate was added to 9 ml of nutrient broth containing 1 mL of OFL (concentrations equal to MIC). Bacterial growth was quantified at time ‘0’ h (immediately after addition of the OFL) and also at defined time intervals (6, 12, 18, 24 and 30 h) by aseptically taking 1 mL of aliquot, diluting serially (tenfold dilutions) in sterile dH20 and plating out 1 mL of the final dilution onto a nutrient agar plate. All plates were incubated aerobically at 37 °C for 24 h and after incubation, the colonies on each plate were enumerated and viable cells were expressed as CFU/mL. The same procedure described above was repeated for LEV and CIP. A growth control comprising the inoculated broth medium without the antibiotics was set up, and 1 mL was plated on nutrient agar. The percent and log reductions of the bacterial cells exposed to OFL, LEV and CIP were calculated for each of the time intervals. The Log10 CFU/mL of survived bacterial cells against exposure time (hrs) were plotted on a semi-logarithm graph for each isolate to obtain time-kill curve. Activity of the antibiotics was considered bacteriocidal at the lowest concentration that reduced the initial inoculum by > 3 log10 CFU/mL (99.9%) and bacteriostatic at the lowest concentration that reduced the initial inoculum by < 3 log10 CFU/mL.

Reductions of the bacterial cells exposed to fluoroquinolone antibiotics

The percentage and logarithm reductions of the bacterial cells exposed to each antibiotic: OFL, LEV and CIP were, respectively, calculated as follows:

$$begin{aligned} & {text{Percentage}};left( % right);{text{reduction}} = left( {frac{{{text{Initial}};{text{counts}}{-}{text{Counts}};{text{at}};text{`}xtext{‘};{text{interval}}}}{{{text{Initial}};{text{counts}}}}} right){text{multiplied}};{text{by}};100 \ & {text{Logarithmic}};left( {{text{Log}}} right);{text{reduction}} = {text{Log}}_{10} left( {{text{Initial}};{text{counts}}} right){-}{text{Log}}_{10} left( {{text{Counts}};{text{at}};text{`}xtext{‘};{text{interval}}} right). \ end{aligned}$$

Statistical analysis

All experiments were performed in triplicates, and statistical significance difference (P < 0.05) between the mean values was determined by Duncan multiple range test using Statistical Package for Social Sciences (SSPS version 22).

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