MATERIALS AND METHODS

Sample collection: The study was conducted among HIV infected individuals attending a Specialist Hospital in Akure, Nigeria, where a total of 12 blood samples were collected from HIV patients on retroviral drugs who also seek medical attention from this selected hospital. About 5 mL of blood samples were collected from the HIV patients by venous puncture into sterile EDTA bottles and transported to the laboratory immediately for bacteriological analyses.

CD4⁺ cell count determination: About 20 μL of the whole blood (EDTA anti-coagulated) was mixed with 20 μL of fluorochrome-labeled antibodies, mixed gently and incubated for 15 min in the dark at room temperature. Afterwards, about 800 μL of lyse buffer was added to the mixture, swirl gently for even distribution and ran on the flow cytometry machine. The CD4⁺ was displayed and recorded.

Bacteriological analyses: Culture media including Nutrient Agar (NA), Cystine Lactose Electrolyte Deficient agar (CLED), MacConkey agar, Blood agar and Chocolate agar were prepared according to manufacturer’s specification and sterilized. The samples were each pour plated on the culture medium and the isolated bacteria were identified and characterized using standard protocols²⁰.

Antibacterial sensitivity test: The isolates were tested for their sensitivity to commercial antibiotics disc using the Kirby Bauer disc diffusion method²¹ on Mueller Hinton agar plate. The commercial antibiotics disc includes Augumentin (25 μg), Gentamycin (10 μg), Tarivid (30 μg), Streptomycin (30 μg), septrin (30 μg), Chloramphenicol (30 μg), Sparfloxacin (10 μg), Ciprofloxacin (10 μg), Amoxicillin (30μg), Pefloxacin (10 μg) for Gram-negative isolates and Erythromycin (10 μg), Septrin (30 μg), Ciprofloxacin (10 μg), Streptomycin (30 μg), Zinacef (20 μg), Amoxicillin (30 μg), Pefloxacin (10 μg), Gentamycin (10 μg), Ampiclox (30 μg), Rocephin (25 μg) for Gram-positive isolates. Zones of inhibition were determined and interpreted²².

Antibacterial activity of essential oils: The bacterial cultures were tested for the inhibitory effect of essential oils of Black seed and Lemon for different concentration (10, 30, 50, 75 and 100 %) using 10% tween-80 as a miscible diluent by using agar well diffusion method²³. The zones of inhibition (mm) were measured with graduated scale after the period of incubation and the result compared with ciprofloxacin which served as a positive control. The MIC and MBC of the essential oils were determined using broth dilution method²⁴. The lowest concentration at which no visible growth occurs in each culture tubes was taken as MIC, while the lowest concentration where no growth occurred on the agar surface is considered as the MBC.

Ethical approval: Ethical approval was obtained from the State Health Research Ethics Committee (SHREC) of Ondo State, Nigeria. Patients consent were sought before the collection of samples.

RESULTS

Percentage occurrence, distribution and their relative CD4⁺ count: A total of 12 samples were obtained from 2 males and 10 females with a percentage occurrence of 16.67 and 83.33%, respectively. About 33.3% of the HIV seropositive patients each had a CD4⁺ T-cell counts of 200-400 and 400-600 cells μL^–1, while the remaining 34% had a CD4⁺ T-cell counts in a normal range, greater than 600 cells μL^–1 as shown in Table 1.

Percentage occurrence of bacterial isolates from samples of HIV positive individuals: Seven bacteria isolated from the samples include: Bacillus cereus, Staphylococcus aureus, Micrococcus luteus, Corynebacterium xerosis, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa. The B. cereus (18%) and P. aeruginosa (20%) had the highest percentage of occurrence in the blood and urine samples, respectively (Table 2).

Antibiogram of bacterial isolates: All the test isolates showed varying degrees of sensitivities and resistance against the antibiotics. Pefloxacin and Amoxicillin were more potent against the Gram-negative isolates while Ciprofloxacin as well as Pefloxacin were effective against the Gram-positive isolates. However, all the Gram-positive isolates were resistant to Zinacef and Amoxicillin while the Gram-negative isolates were sensitive to at least one of the antibiotics. Both Pefloxacin and Ciprofloxacin displayed a broad spectrum of activity against all the isolates as shown Table 3.

Table 1:	Distribution and CD4+ count of HIV positive individuals

Table 2:	Percentage occurrence of bacterial isolates from the blood and urine of HIV seropositive individuals

Table 3:	Antibiotics sensitivity profile of Gram-negative and Gram-positive bacterial isolates from HIV positive blood and urine samples
Values are mean zone of inhibition (mm) ± Standard deviation of-three replicate, a-cMeans in the same column not sharing a common letter are significantly different (p<0.05) by Duncan's multiple range test

Potency of black seed and lemon oil against the bacterial isolates: The natural compounds compared favorable well as compared with the commercial antibiotics used as a positive control (Ciprofloxacin) as shown in Table 4.

Table 4:	Antibacterial activity of lemon oil and black seed oil on bacterial isolates from blood and urine of HIV seropositive samples
Vales are mean zone of inhibition (mm) ± Standard deviation of three replicate, a-cMeans in the same column not sharing a common letter are significantly different (p<0.05) by Duncan's multiple range test

Table 5:	Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of lemon oil and black seed oil on the bacterial isolates

Both oils were effective at 10% concentrations on all the bacterial isolates. Black seed oil was observed to possess higher antibacterial activity against the isolates as compared to lemon oil, with higher zones of inhibition values of above 13.00±0.40^d mm, which was the highest observed on B. cereus with lemon oil. The B. cereus, P. aeruginosa and M. luteus were mostly sensitive to lemon oil up to 75% concentration while S. aureus and C. xerosis were least sensitive at 30% concentration. However, black seed oil showed potent activity against C. xerosis with the highest zone of inhibition value of 18.67±0.88^e mm and least effective against K. pneumoniae. The MIC and MBC values of the oils ranged from 10-100% as shown in Table 5. The isolates were found to be inhibited by lemon oil at a lower concentration compared to the black seed oil.

DISCUSSION

In this study, a total of 12 HIV seropositive individual’s blood and urine samples were analyzed microbiologically and 7 bacterial isolates were identified, including Staphylococcus aureus, Bacillus cereus, Micrococcus luteus, Corynebacterium xerosis, Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae.

The high vulnerability of HIV infected females compared to males observed in this study might be associated with social, legal and economic factors¹⁹. None of the patients in this study had a CD4⁺ cell count of less than 200 cells μL^–1, moreover, the prevalence of bacteremia amongst HIV positive individuals have been linked with a CD4⁺ T-cell counts below 200 cells μL^–1 which can pose a greater risk of opportunistic bacterial infection¹⁹. This showed the relevance of anti-retroviral treatment of HIV patients with relatively low CD4⁺ count.

The bacterial isolates exhibited significant levels of multidrug resistant. The HIV seropositive individuals are prone and at high risk of bacterial opportunistic infections²⁵. Colonization with S. aureus has been reported to be a risk factor for clinical infections in HIV positive patients^26,27. The S. aureus has been identified as the most common uropathogen²⁸. The occurrence of P. mirabilis agrees with a study on bloodstream infections amongst HIV patients under HAART in Thailand, who linked the occurrence of this organism to complicated urinary tract infections²⁹. C. xerosis has previously been associated with clinical samples linked with animals^30,31. Thus, the immunocompromised status of the host as a result of HIV infection gives a lot of serious concern due to an increase in multidrug resistant pathogens. The sensitivity of the isolates to Ciprofloxacin and Pefloxacin implies the effectiveness of quilonones class of antibiotics to opportunistic bacterial pathogens. The inappropriate usage of broad spectrum antibiotics might account for the multidrug resistant pattern observed in the isolates to the commercial antibiotics³. Lemon oil possess antimicrobial compounds such as limonene, phenols, terpenes and aromatic compounds, which enhances their antibacterial property³². Phenolic compounds of essential oils have been shown to lead to cell membrane disruption leading to leakage of cellular constituent³³. Black seed oils are also rich sources of antimicrobial compounds such as thymoquinone and hydrothymoquinone³⁴. Gram-positive bacteria appears to be more susceptible to essential oils compared to Gram-negative bacteria due to the nature of their cell wall. Black seed oil is reported to have strong antibacterial activity against some selected Gram-positive and Gram-negative bacterial species³⁵.

Antibiotics have been the most frequently prescribed medications in modern medicine and has led to various immunocompromised conditions of the host immunity. These essential oils have been shown to possess therapeutic values however, further research is needed to study their efficacy in vivo.

CONCLUSION

Quinolones such as ciprofloxacin and Pefloxacin showed better sensitivity and broad spectrum pattern to the bacterial isolates from HIV seropositive samples, however essential oils such as lemon and black seed oil compared favourably well with these antibiotics. Moreover, Black seed oil had better antibacterial activity compared to lemon oil. Essential oils can serve as potential candidates against opportunistic bacterial pathogens associated with immunocompromised conditions such as HIV.

SIGNIFICANCE STATEMENT

This study has provided information on some pathogenic bacteria that are associated with urine and blood samples of HIV seropositive patients. The antibiotics sensitivity profile also revealed the multi-drug resistant pattern of these isolated bacteria and the antibacterial efficacy of black seed and lemon oil has also been demonstrated. This will give a base line information on a possible alternative therapy towards eradicating opportunistic bacterial infections commonly associated with this viral disease.