Introduction

Ceftazidime belongs to cephalosporin class of antibiotics with broad spectrum activity.^{1, 2} It is stable to both plasmid and chromosomal β – lactamase resistance then other cephalosporins.^{3, 4} Ceftazidime is a third generation cephalosporin and is resistant to hydrolysis. It is effective against a broad range of gram positive and gram negative bacteria and also against bacteria resistant to cephalosporins.

Tobramycin is an aminoglycoside antibiotic used to treat various types of bacterial infections, particularly gram  negative infections. It  is often used concomitantly with other antibacterials to extend its spectrum of efficacy or increases its effectiveness. Treatment with a combination of an aminoglycoside  with  a  β – lactam has showed  increased efficacy.

Ceftazidime and tobramycin combination therapy is considered by some clinicians to be the clinical standard.⁵ Antibacterial drugs have been highly successful in controlling the morbidity and mortality that accompany serious bacterial infections. Some of the exiting antibiotics may cause adverse effects in some patients. Some of  these side effect  may be significant enough to require that therapy should be discontinued.^{6, 7}

Combination therapy of cephalosporins and aminoglycosides is also used to broaden the antimicrobial spectrum in critically ill patients while awaiting a bacteriological diagnosis or  proven polymicrobial infection. Synergism appears to be maintained even at very  high  MIC  with drug  combinations within achievable therapeutic ranges.^{8, 9, 10, 11}

The fight against bacterial infection represents one of the highest point of the modern medicine. Since the development of  antibiotics, this powerful tool has saved millions of  lives. However, because of inappropriate and large use of antibiotics, many antibiotic resistant strains are growing in number. The resistant bacteria pose a significant threat to human health and a challenge to researches.^{12, 13} Keeping this in the view, the present study was planned  to evaluate efficacy  of  Tobracef, FDC of ceftazidime and tobramycin against some clinically significant microorganisms.

Materials and Methods

Bacterial Strains

Following strains obtained from Microbial Type Collection Center of Institute of Microbial Technology, Chandigarh, India were  used for the study, Acinetobacter baumanii (MTCC No. – 1425), Mycobacterium smegmatis (MTCC No. – 995), Citrobacter braaki (MTCC No. – 2690) and Pseudomonas aeruginosa (MTCC No. – 1688)

Antibiotic

Tobracef, ceftazidime and tobramycin used in study were provided by manufacturer, Venus Remedies Limited, India.

Medium

Mueller Hinton (MH) media supplemented with Calcium (25 mg/l) and Magnesium (1.25 mg/l) was used for MIC and susceptibility tests experiments. Colony counts were determined with MH agar plates.

Susceptibility Testing

The MIC of ceftazidime and tobramycin alone and in a Tobracef  against A. baumanii, M. smegmatis, C. braaki and P. aeruginosa were determined by broth micro dilution method as per the standard National Committee for Clinical Laboratory Standards.¹⁴ Overnight MH broth cultures were used to prepare inocula of  10⁵  CFU/ml. The MIC was defined as the lowest concentration of antimicrobial  agent that prevented  turbidity after 24 hours of  incubation at 37 ⁰C.

Results

MIC studies

In case  of A. baumanii, M. smegmatis, C. braaki and P. aeruginosa MIC were found to be 0.25 mg/l,  0.25 mg/l, 1 mg/l and 1 mg/l for ceftazidime respectively and in tobramycin  alone the  MIC  were found to be  0.125 mg/l, 0.25 mg/l, 0.0625 mg/l and 0.5 mg/l respectively. In a tobracef  MIC were found to be  0.0625 mg/l, 0.125 mg/l, 0.03125mg/l and 0.25 mg/l respectively.

The MIC of all microbial strains under study resulted in significant reduction in ceftazidime, tobramycin  alone  and tobracef. (Table -1)

Table 1: Minimum Inhibitory Concentrations determination of  ceftazidime, tobramycin  and Tobracef  with  A. baumanii, M. smegmatis, C. braaki and P. aeruginosa.

Time kill curve analysis

Bactericidal effect, with 2X the MIC of tobracef, ceftazidime and tobramycin achieved the earliest killing at 4 hours. Bacterial killing rate in toracef were distinctly higher than ceftazidime and tobramycin  alone in all the strains under study. (Fig – 1, Fig – 2, Fig – 3 and Fig – 4).

In  A. baumanii  time kill curve analysis demonstrated bacterial killing in 0 hrs, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs and 12 hrs for ceftazidime alone 6.31 log₁₀CFU/ml, 5.94 log₁₀CFU/ml, 4.95 log₁₀CFU/ml, 5.40 log₁₀CFU/ml, 5.62 log₁₀CFU/ml, 6.27 log₁₀CFU/ml and 6.46 log₁₀CFU/ml, for a  tobramycin alone 6.15 log₁₀CFU/ml, 5.81 log₁₀CFU/ml, 4.70 log₁₀CFU/ml, 5.32 log₁₀CFU/ml, 5.76 log₁₀CFU/ml, 6.26 log₁₀CFU/ml and 6.40 log₁₀CFU/ml, and tobracef were 6.09 log₁₀CFU/ml, 5.76 log₁₀CFU/ml, 4.30 log₁₀CFU/ml, 5.08 log₁₀CFU/ml, 5.59 log₁₀CFU/ml, 6.09 log₁₀CFU/ml and 6.33 log₁₀CFU/ml. (Fig – 1)

Figure 1   Click here to View figure

In M. smegmatis time kill curve analysis demonstrated bacterial killing in 0 hrs, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs and 12 hrs for tobracef were 5.99 log₁₀CFU/ml, 5.43 log₁₀CFU/ml, 4.30 log₁₀CFU/ml, 5.08 log₁₀CFU/ml, 5.48 log₁₀CFU/ml, 5.92 log₁₀CFU/ml and 6.10 log₁₀CFU/ml respectively, for ceftazidime alone 6.25 log₁₀CFU/ml, 5.49 log₁₀CFU/ml, 4.48 log₁₀CFU/ml, 5.18 log₁₀CFU/ml, 5.48 log₁₀CFU/ml, 5.96 log₁₀CFU/ml and 6.16 log₁₀CFU/ml receptively and tobramycin alone 6.01 log₁₀CFU/ml, 5.49 log₁₀CFU/ml, 4.48 log₁₀CFU/ml, 5.18 log₁₀CFU/ml, 5.48 log₁₀CFU/ml, 5.96 log₁₀CFU/ml and 6.10 log₁₀CFU/ml. (Fig –  2)

Figure 2: Time Kill Curve of  M. smagmatis.   Click here to View figure

In  C. braaki  time kill curve analysis demonstrated bacterial killing in 0 hrs, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs and 12 hrs for ceftazidime alone 6.33 log₁₀CFU/ml, 5.99 log₁₀CFU/ml, 4.90 log₁₀CFU/ml, 5.46 log₁₀CFU/ml, 5.83 log₁₀CFU/ml, 6.04 log₁₀CFU/ml and 6.29 log₁₀CFU/ml, for a  tobramycin alone 6.22 log₁₀CFU/ml, 6.00 log₁₀CFU/ml, 4.85 log₁₀CFU/ml, 5.54 log₁₀CFU/ml, 5.77 log₁₀CFU/ml, 6.07 log₁₀CFU/ml and 6.32 log₁₀CFU/ml, and tobracef were 6.11 log₁₀CFU/ml, 5.98 log₁₀CFU/ml, 4.70 log₁₀CFU/ml, 5.45 log₁₀CFU/ml, 5.65 log₁₀CFU/ml, 6.02 log₁₀CFU/ml and 6.27 log₁₀CFU/ml. (Fig – 3)

Figure 3: Time Kill Curve of  C. braakii.   Click here to View figure

In P. aeruginosa time kill curve analysis demonstrated bacterial killing in 0 hrs, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs and 12 hrs for tobracef were 6.46 log₁₀CFU/ml, 6.26 log₁₀CFU/ml, 4.78 log₁₀CFU/ml, 5.96 log₁₀CFU/ml, 6.26 log₁₀CFU/ml, 6.46 log₁₀CFU/ml and 6.55 log₁₀CFU/ml respectively, for ceftazidime alone 6.47 log₁₀CFU/ml, 6.28 log₁₀CFU/ml, 5.00 log₁₀CFU/ml, 5.80 log₁₀CFU/ml, 6.12 log₁₀CFU/ml, 6.48 log₁₀CFU/ml and 6.60 log₁₀CFU/ml receptively and tobramycin alone 6.45 log₁₀CFU/ml, 6.27 log₁₀CFU/ml, 4.95 log₁₀CFU/ml, 6.02 log₁₀CFU/ml, 6.28 log₁₀CFU/ml, 6.47 log₁₀CFU/ml and 6.55 log₁₀CFU/ml. (Fig –  4).

Figure 4: Time Kill Curve of  P.  aeruginosa.   Click here to View figure

Discussion

A further indication for antibiotic combinations is to prevent emergence of resistance.¹⁵ A synergistic interaction between the two antibiotics is one reason for using this combination.¹⁶ Antibiotic combinations have long been used to provide antibacterial activity against  multiple potential pathogen for initial empirical treatment of critically ill patients. Several studies of antibiotic combination therapy  for gram negative infection conducted from the 1970s  to the 1990s. The consensus  is that combination therapy is probably more effective than mono therapy only for infections. Gram negative bacterial species typically have a higher degree of antibiotic resistance than gram positive bacteria. This is largely in part due to the presence of a selectively permeable outer membrane which restricts the entrance of small hydrophobic molecules, including many available antibiotics.¹⁷  Aminoglycoside class antibiotic exert a killing effect by binding to bacterial ribosomes and inhibiting bacterial protein synthesis.

Ceftazidime is a third generation cephalosporins, with good antibacterial activity.^{2, 18} Clinically administration of  two or more antibiotics in the treatment of infections is usually rationalized with the knowledge that multiple antibiotics often exert additive or synergistic effects, increasing the likelihood of pathogen eradication. In comparison with older cephalosporins, it crosses the bacterial outer cell membrane faster and has advantages of rapid penetration in periplasmic space as well as of extended spectrum of  the activity that include gram positive and gram negative organisms.

Useful antibiotic classes based  on a β – lactam structure include broad  penicillins, cephalosporins, carbapenems, all of which inhibit bacterial cell wall synthesis. β – lactamase enzymes that rapidly degrade the cephalosporins  β – lactam ring have a primary bacterial resistance mechanism  against this class of drug since the commencement of clinical cephalosporins use in the 1940s. Because of  this, most cephalosporins derived antibiotics still clinically used  are formulated to include a  β – lactamase  inhibitor  in order to increase the drug’s effectiveness. Cephalosporins also contain a  β – lactam ring, but are structurally more resistance  to  β – lactamase  degradation.

Tobracef  has lower MIC than ceftazidime and tobramycin alone against A. baumanii, M. smegmatis, C. braaki  and P. aeruginosa.

This investigation indicated that Tobracef has better efficacy as compared to ceftazidime and tobramycin alone  in organisms under  study.

Acknowledgments

Authors are greatful to acknowledge the CGM, Domestic operations of Venus Remedies Limited for providing the samples of  ceftazidime, tobramycin and Tobraceffor the study and financial support for this investigation.

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