The Interaction Between Gentamicin and Floroquinolones Against ESBL Producing Clinical Isolates of Escherichia coli

MATERIALS AND METHODS

Sample Collection
One hundred and four clinical isolates of E. coli were collected from the intensive care unit of Eastern Nigeria Medical Centre Enugu. The isolates were obtained from urine (26), blood (24), stool (20), sputum (19) and serum (14). They were subsequently identified and characterized using standard microbiology techniques (Chessbrough, 2002).

Culture Media
Mueller-Hinton Agar (Oxoid UK) and Nutrient Broth (Fluka) were prepared according to manufacturer`s specifications.

Susceptibility Studies
This was conducted using the disc diffusion method as described by Bauer et al. (1966). Antibiotics tested were gentamicin (25 μg), nalidixic acid (25 μg), ceporex (30 μg), ofloxacin (25 μg), cipofloxacin (25 μg), perfloxacin (25 μg), ampicillin (25 μg), septrin (25 μg), amikacin (25 μg), ceftazidime (30 μg), cefotazime (30 μg), ceftriaxone (30 μg) and imipenem (30 μg).

Double Disc Synergy Test
Test isolates showing resistance to any of the 2nd and 3rd generation cephalosporins were subjected to double disc synergy test studies. Individual discs containing 30 μg of ceftazidime, ceftriaxone and cefotaxime were placed on the plate at a distance of 15 mm (edge to edge) from an amoxicillin/clavulanic acid disc (20-10 μg) placed at the center of the plate. An enhanced zone of inhibition between any of the β-lactamase discs and the disc containing clavulanic acid was interpreted as a positive result.

Preparation of Drug Stock Solution
A stock solution of gentamicin was prepared by dissolving in appropriate quantity of sterile distilled water to get 10,000 μg mL^-1 and stock solutions of perfloxacin, ciprofloxacin, ofloxacin and gatifloxacin were dissolved in appropriated quantity of sterile distilled water to get 5,000 μg mL^-1. Seven different 2-fold arithmetical serial dilutions were made with each drug at their stock concentration.

Interaction Studies
Stock solutions of gentamicin (10,000 μg mL^-1), ciprofloxacin, ofloxacin, gatifloxacin and perfloxacin (5,000 μg mL^-1) were freshly prepared for the evaluation of their combined effects against five representative ESBL positive E. coli isolate from each specimen. The two agents were mixed in varying ratios ranging from 0:10 gentamicin to ciprofloxacin, perfloxacin, ofloxacin and gatifloxacin and 10:0 of same agents in accordance with the continuous variation checkerboard protocol (Esimone et al., 1999; Okore, 1990). Each of the eleven combinations of these five agents was serially diluted (2-fold) with sterile distilled water. One milliliter of each of the drug combinations was respectively seeded in a Petri dish together with 19 mL of sterile Mueller Hinton agar and allowed to stand for 1 h to solidify and for pre-diffusion of the drugs. An aliquot equivalent to 0.5 Macfarland standard of each selected ESBL producing E. coli was streaked on the surface of the agar plates. The set up was conducted in triplicates with a control containing no antibiotics. They were then incubated at 37°C for 24 h. The MICs of the various combination proportions were determined and interactions between the antimicrobial agents were accessed by determining their fractional inhibitory concentrations (FIC) index according to the relationship.

FIC index = FIC_A+FIC_B where A and B are two antimicrobial agents being combined.
FIC_A = (MIC of drug A in combination with drug B)/(MIC of drug A alone)
FIC_B = (MIC of drug B in combination with drug A)/(MIC of drug B alone)
The activity index (AI) = Log FIC index.

RESULTS AND DISCUSSION

The result of the study revealed that 15 (14.42%) out of 104 clinical isolates of E. coli screened were positive for ESBL enzyme production. The highest frequency occurred in urine specimen 4 (3.8%) while the least occurred in semen 2(1.9%). The ESBL enzyme present is predominantly of SHV and TEM type. This was inferred from the fact that ceftazidime was the antibiotic that showed increase in the zone of inhibition above 5 mm towards the combination disc (amoxicillin/clavulanic acid).

The combination interaction studies showed that some ratios of antibiotic combinations were synergistic while some were additive, antagonistic and indifference in activity, respectively. The combinations of gentamicin and gatifloxacin, ciprofloxacin and perfloxacin were predominantly synergistic (Table 1-3), while that with ofloxacin were mainly indifference (Table 4). The combination ratio 8:2 produced the highest synergy (80%) for gatifloxacin combination and 3:7 and 9:1 for ciprofloxacin combination. A (100%) synergy was recorded for perfloxacin combinations at the ratio of 1:9 and 6:4. However, the highest indifference activity of ofloxacin combination was obtained at 2:8 ratio.

Table 1:	Activity of gentamicin and gatifloxacin against five ESBL producers by checkerboard method

Table 2:	Combined activity of gentamicin ant ciprofloxacin against five ESBL producers by checkerboard method

Table 3:	Combined activity of gentamicin and perfloxacin against five ESBL producers by checkerboard method

Table 4:	Combined activity of Gentamicin ant ofloxacin against five ESBL producers by checkerboard method

ESBL producing organisms are known to be resistant to beta lactam antibiotics. This is because ESBL producers have enzymes with relaxed active site that can encompass the beta lactam with large side groups protecting the beta lactam ring, thus making these organisms resistant to virtually all beta lactam antibiotics. This enzyme have spread world wide with intra and inter-species transfer being facilitated by plasmid encoded enzymes. The present study indicated 14.4% prevalence of ESBL producing Escherichia coli in Enugu, Nigeria. This figure is slightly less than the 16.5% prevalence reported from Abakaliki, Nigeria (Iroha et al., 2008). The recovery of ESBL producing E. coli in Enugu, Nigeria could pose a lot of clinical challenges especially in recent times when antibiotics other than the beta lactams are reported to develop resistance (Shannon and French, 2004). This later development is worrisome, since the aminoglycosides , floroquinolones and cabapenems were hitherto recommended as alternative to cephalosporins for ESBL producing organisms (Aibinu et al., 2003). In this connection, the need to search for effective substitute remedy against ESBL producers remains a major challenge to scientists.

The result of the interaction study in the present study indicated the predominance of synergy in the combinations of gentamicin and ciprofloxacin, perfloxacin and gatifloxacin. Hundred percent synergy was recorded for the gentamicin/perfloxacin combination at the ratios of 1:9 and 6:4 (Table 4). This discovery is very gratifying and brightens the hope for the remedy search for ESBL enzyme producers.

The synergistic interaction between gentamicin and ciprofloxacin recorded in this study is in line with the report of Mandal et al. (2003), in which combination of the two antibiotics produced enhanced activity against ESBL producing enteric organism. Further, a study carried out in the United States established that gatifloxacin was synergistic with beta lactams including piperacillin, cefepime and meropenem and with gentamicin, against some drug resistant pathogens (Dawis et al., 2003). It is undoubtable from the result of the present study, that drug combination could be the longed-for panacea needed for the management of ESBL producing strains of E. coli and perhaps other organisms. A further study on the drug interaction against other ESBL producing organisms is hereby emphasized. In conclusion, the enhanced in vitro activity resulting from antibiotic combinations in this study could be of immense significance in the successful treatment of most fatal bacterial diseases. This finding is particularly vital in the developing nations, where single effective drugs against ESBL producing organisms are either expensive or unavailable. Consequently, the establishment of such drug combinations for all the common ESBL producing microorganisms is at present, a compelling necessity, if a successful battle against ESBL producers will be achieved.