|Year : 1998 | Volume
| Issue : 2 | Page : 97-101
Microbiological assay of ampicillin in serum and aqueous humor of patients given ampicillin-sulbactam injection
HN Madhavan, A Biswas, J Malathy, BN Mukesh
Vision Research Foundation, Chennai, India
H N Madhavan
Vision Research Foundation, Chennai
Source of Support: None, Conflict of Interest: None
The aim of this study was to determine the bacterial growth inhibitory activities of ampicillin in aqueous humor and serum of patients administered ampicillin-sulbactam combination intramuscularly prior to cataract surgery. 43 patients received a combination of both antibiotics intramuscularly at varying periods (60-140 minutes) prior to surgery. Aqueous humor and venous blood were collected at the beginning of the surgery. For microbiological assay, spores of Bacillus subtilis were incorporated in the agar. The test sample and the standard solutions (calibrators) of ampicillin and ampicillin-sulbactam combination were placed in 3 mm wells in the agar. The diameter zones of growth inhibitory activities of ampicillin of the calibrators and the test samples measured in mm were extrapolated to the standard curve and were recorded as ampicillin activity in (μg/ml. The results of the assay were placed in 5 groups according to the time intervals between injection and collection of serum and aqueous humor (≤70, 75, 80, 90, >90 minutes). Ampicillin activities in sera and aqueous humor of group 5 (>90 minutes) were significantly higher than the others (p<0.001). The ratio of ampicillin activities of sera and aqueous humor in group 5 patients was significantly lower indicating higher concentration of ampicillin activity in aqueous humor during this period. Bacterial growth inhibitory activities of ampicillin-sulbactam combination were adequate in aqueous humor of all patients with highest activity being 90 minutes after intramuscular administration indicating the potential usefulness of this antibiotic combination as chemoprophylaxis prior to cataract surgery.
Keywords: Microbiological assay, ampicillin, sulbactam, aqueous humor, serum
|How to cite this article:|
Madhavan H N, Biswas A, Malathy J, Mukesh B N. Microbiological assay of ampicillin in serum and aqueous humor of patients given ampicillin-sulbactam injection. Indian J Ophthalmol 1998;46:97-101
Postoperative endophthalmitis is one of the most feared complications of cataract surgery. In a bacteriological study on anterior chamber aspirates collected at the end of uncomplicated cataract surgery from 30 patients, 43% were bacterial culture positive, but none developed endophthalmitis probably due to sub-conjunctival inoculation of gentamycin. In attempts to prevent postoperative endophthalmitis, ophthalmologists often use topical or systemic antibiotics prior to surgery. For this to be successful the antibiotic administered prior to surgery should be present in sufficient amount in the anterior chamber to inhibit or destroy the bacteria which enter into it at the time of surgery. Adequate concentrations of the antibacterial drugs in aqueous humor have been shown to be present on systemic administration of rifampicin and ciprofloxacin but not so for cefotaxime. Topical administration of norfloxacin results in its penetration into anterior chamber in adequate concentration.
In our hospital the use of ampicillin as a prophylactic in cataract surgery is a policy unless otherwise indicated and is administered 1-2 hours prior to surgery. Ampicillin in combination with β-lactamase inhibitor sulbactam is effective against bacteria that have acquired resistance to ampicillin., Both antibiotics when administered as intravenous infusion have favourable pharmacokinetics and penetration into lung tissue, tissues during otolaryngology surgery, blister fluids, and tissues in diabetes related foot infections.
As there was a policy decision being taken to introduce ampicillin-sulbactum combination as the prophylactic in our hospital, the present study was undertaken to determine the bacterial growth inhibitory activity of ampicillin in combination with sulbactam in anterior chamber of the eye in patients administered with the above antibiotics combination prior to cataract surgery.
| Materials and Methods|| |
| Patients and specimens|| |
43 patients ranging in age from 40 to 85 years, undergoing planned cataract extraction with intraocular lens implantation, were included in this study. There were 20 males and 23 females. Patients were chosen randomly to receive the combination of ampicillin (1 gm) and sulbactam (0.5 gm) intramuscularly (IM) 60-140 minutes (the period between administration of the antibiotic combination and collection of samples) prior to surgery. Aqueous humor was aspirated using 26 gauge needle attached to a tuberculin syringe at the beginning of the cataract surgery. Within 5 minutes, 5 ml of venous blood was collected in a sterile dry blood collection test tube and both were immediately sent to laboratory where serum separated from the venous blood and aqueous humor were stored at -20° C until both were processed.
| Microbiological assay|| |
Agar diffusion method of microbiological assay was carried out. Microbiological assay plate containing Bacillus subtilis spores and standard solutions (calibrators) of ampicillin and ampicillin-sulbactam combination were prepared as follows.
| Preparation of microbiological assay plate|| |
To 100 ml of sterile, melted and cooled (about 45° C) Mueller-Hinton agar medium, 0.2-0.4 ml of spore suspension of Bacillus subtilis var niger (ATCC 6644) containing approximately 6X104 colony forming units (CFU) per ml, was added and mixed well without any air bubble formation. 20 ml of this medium was poured into 100 mm sterile glass petri dish (Borosil, India). As soon as the medium was solidified, 9 wells of 3 mm diameter were cut in it as shown in [Figure:1] and [Figure:2]. The plates were stored at 4° C until used within 48 hours after its preparation.
| Preparation of ampicillin assay calibrators|| |
Ampicillin pure powder (FDC, India) was dissolved in 0.1 M phosphate buffered saline (PBS) to provide a stock solution of 2.56 mg/ml. Serum calibrators: Pooled human serum was used as diluent for dilution of stock ampicillin to obtain 4 calibrator solutions containing 400, 100, 20 and 4 μg/ml. Aqueous humor calibrators: 0.1 M PBS was supplemented with 2% pooled human serum to mimic protein concentration of aqueous humor (50 mg/dl). Ampicillin stock solution was diluted in the above solution to obtain 4 calibrator solutions containing concentrations of 40, 20, 10 and 5μg/ml (2 fold dilutions).
| Preparation of ampicillin-sulbactam assay calibrators|| |
A stock solution of 2.56 mg/ml of sulbactam was prepared by dissolving pure powder of ampicillin in 0.1 M PBS. Serum and aqueous humor calibrator solutions of both antibiotics combination were prepared from stock solutions in proportions of 2:1 of ampicillin and sulbactam maintaining the same concentrations of ampicillin as in its calibration solutions.
Pooled human serum required as diluent of the antibiotics was prepared from sera aseptically collected from blood obtained from 4-6 healthy human volunteers and sterilized by membrane filtration. Whole pooled human serum was used as diluent for the calibrators to be equivalent in nature to the serum sample to be tested, and similarly, 0.1 M PBS was supplemented with 2% pooled human serum to mimic protein concentration of aqueous humor (50 mg/dl) for it to be equivalent to aqueous humor to be tested. The pooled sera were pre-tested for any antibacterial activity in the microbiological assay plate and only those free of any such activity were used in the assay.
| Performance of microbiological assay|| |
This was carried out within 24-48 hours of collection of samples. One microbiological assay plate was used for one test sample. Calibrators prepared for serum and aqueous humor were used in the assay. 50 μl each of the 4 calibrators for ampicillin and 4 for ampicillin-sulbactam combination were placed in the 8 peripheral wells and the test sample in the center well of the microbiological assay plate [Figure:1] and [Figure:2] which was incubated at 25° C for 16-18 hours. The zone of inhibition in diameter was determined to the nearest mm using zone reader. While designing the assay the indicator bacterium (B. subtilis var niger ATCC 6644), medium (Mueller-Hinton agar, HiMedia, India), sample size (50 μ1), and incubation temperature (25° C) were optimized to give a suitable zone diameter of inhibition of growth for the concentrations of the calibrators used in the assay. Bacillus subtilis spore suspension was chosen specifically to maintain approximately the same number of viable bacteria throughout the experimental period.
| Calibration|| |
The zone of inhibition of bacterial growth in diameter around the calibrators and the test samples (serum and aqueous humor) was determined to the nearest mm using a zone reader. The diameters of inhibition of bacterial growth of the calibrators of ampicillin and ampicillin-sulbactam combinations were 2-3 mm more than those of ampicillin alone indicating the enhanced activity of ampicillin in the presence of sulbactam. The bacterial inhibitory activity of ampicillin was recorded in μg/ml used in each well of the calibrators. The relationship of diameter zones of inhibition for the 4 concentrations of the calibrators of ampicillin-sulbactam combination and log concentration of ampicillin of each of these 4 calibrators was obtained as linear. Using the diameter zone of inhibition of the test sample (serum or aqueous humor), its ampicillin activity was calculated interpolating it with the standard curve derived by the method of least squared procedure as shown in [Figure:3] and [Figure:4].
| Results|| |
[Table:1] shows the results of microbiological assay of ampicillin activity in sera and aqueous humor of patients according to the time interval of their collection after IM injection of ampicillin-sulbactam combination. Ampicillin activity in serum varied from 1.39 to 33.11 μg/ml and its concentration in aqueous humor varied from 0.02 to 25.59 µg/ml. In 4 patients, the ampicillin activity was more in aqueous humor than in their respective serum [Table:1].
For the purposes of statistical analysis, the results of the assay were placed in 5 groups according to the time intervals between IM injection and collection of serum and aqueous humor samples as given in [Table:2].
The median values of ampicillin activities of ampicillin-sulbactam combination in serum and aqueous humor and the ratios of these values for the respective groups are shown in [Table:2]. Kruskal-Wallis nonparametric analysis of variance was applied to compare the ampicillin activity in serum and aqueous humor among these 5 groups. The median values of its activity varied from 4.77 to 16.64μg/ml in serum and from 0.45 to 10.31μg/ml in aqueous humor. Sera and aqueous humor of patients showed significant differences of ampicillin activity among 5 groups both in serum (p=0.008) and in aqueous humor (p<0.001).
By performing Dunn's multiple comparisons procedure for ranks, significant differences in ampicillin activity in sera (p<0.001) were observed between the groups 1-4 and 5, and its activity was significantly higher in aqueous humor (p<0.001) of group 5 than the others. Mann-Whitney U test also showed the ratio of ampicillin activity of sera and aqueous humor of group 5 patients (whose test samples were collected 90 minutes after IM injection of the antibiotics) was 1.95, which is significantly lower (p=0.019) when compared to those from patients of groups 1, 2, 3, and 4, their median ratio value being 4.34.
| Discussion|| |
Ampicillin, an aminopencillin, has a broad spectrum of activity against both gram positive and gram negative bacteria. The minimum inhibitory concentrations (MIC) for sensitive gram positive bacteria is reported to range from 0.02 to 6 μg/ml and for gram negative bacteria from 0.02 to 8 μg/ml. Sulbactam is a broad spectrum irreversible inhibitor of β-lactamases. In combination with sulbactam, effectiveness of ampicillin activity against resistant bacteria is restored and antibacterial spectrum is also extended to cover species that have never been susceptible to ampicillin.
Our study has clearly demonstrated the presence of adequate (more than the required MIC) growth inhibitory activity of ampicillin in combination with sulbactam in serum and aqueous humor during the period of 60-140 minutes after an IM injection of the antibiotic combination with the highest activity being after 90 minutes. In an earlier study, using reverse phase high performance liquid chromatography, we have demonstrated adequate concentrations of ampicillin and sulbactam in sera and aqueous humor samples of 25 patients under similar conditions. The bacterial growth inhibitory activities of sera and aqueous humor test samples in this study were reflective of biological effectiveness of the presence of both antibiotics. During routine intraocular surgery, fluid from conjunctival sac contaminated with resident bacteria enters into the anterior chamber, and may cause endophthalmitis. Several of them are known to produce β-lactamases naturally. Ampicillin alone used for prophylaxis prior to intraocular surgery may not be effective against invasion of such organisms, particularly several gram negative bacilli. Ampicillin with sulbactam combination may be more useful.
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