Antibiotic combinatorial approach utilized against extended spectrum beta-lactamase ( ESBL ) bacteria isolates from Enugu , South Eastern Nigeria

© 2014 Ruth A. Afunwa et al.; licensee University of Sarajevo Faculty of Health Studies. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. UNIVERSITY OF SARAJEVO FACULTY OF HEALTH STUDIES ABSTRACT


INTRODUCTION
Several community-acquired pathogens that commonly cause diarrhoea have been found to be ESBL producers.In the last 3 years there have been reports of true community-acquired infection or colonization with ESBL-producing Escherichia coli from Spain, Israel, the United Kingdom, Canada, and Tanzania (1)(2)(3)(4).Typically, patients have developed urinary tract infection with CTX-M-producing Escherichia coli.Some urinary tract infections have been associated with bacteraemia.Th e majority of isolates have been resistant to commonly used fi rst-line agents for urinary tract infection such as trimethoprim-sulfamethoxazole, ciprofl oxacin, gentamicin, and ceftriaxone.Th e cause of this sudden upsurge in community-acquired infections with ESBL-producing organisms is not yet clear, but associations with foodstuff s, animal consumption of antibiotics, and frequent patient contact with health care facilities need to be explored.Th e ESBL production status of the isolates were previously established through the double disc synergy test (DDST) method involving the use of a combination disc (Amoxicillin 20 μg and Clavulanic acid 10 μg) placed at the centre of the Petri dish and antibiotics (Ciprofl oxacin 30 μg and Cefuroxime 30 μg) placed 15 mm apart on both sides of the plates (5).Also, in our report we could establish their community or nosocomial origins (5).Th e purpose of this present work was to study some commonly applied antibiotic in quite various novel combinations for use against ESBL-producing bacteria isolates.In this paper, we present the result of our antibiotic susceptibility studies of the ESBL isolates to a cocktail of antibiotics to establish a very realistic antibiogram that may prove useful in clinical practice.Th e synergies that may exist between the antibiotics involved in each combination were analyzed.

Microorganisms
From a total of 460 samples collected from volunteers and patients (over a fi ve months period between October 2006 and February 2007) after informed consent and ethical approval, 20 ESBL producers were identifi ed.From these 8 were recruited into the antimicrobial combinatorial therapeutic studies.Th e samples were processed through the following hospitals: respectively from four hospitals comprising University of Nigeria Teaching Hospital (UNTH); Enugu, National Orthopaedic Hospital, Enugu (NOHE), Ntasiobi Ndinona Afufu (NONA), and Reego Laboratories, Enugu.Characterization of isolates was according to recommended standard technique by the National Committee for Clinical Laboratory Standard (NCCLS).

Antibiotic Sensitivity test
Antibiotic sensitivity of the isolates was determined using previously established procedure (6).Briefl y, the isolates were cultured in nutrient broth at 370C for 24 h.Two loopfuls of the suspension of each isolate were inoculated into 20ml of sterile molten agar in 10 cm diameter Petri dishes and mixed.Th e plates were allowed to set and the antibiotic Sensitivity discs were aseptically placed on their surfaces.Th e plates were incubated at 370C for 24 h and the resultant inhibition zone diameters (IZDs) were measured and recorded.

Combined Activity of Gentamicin with other Antibiotics
A 1000 μg/ml stock solution of gentamicin and 5000 μg/ml each of Augmentin® (Amoxicillin and clavulanic acid), cefoxitin, ceftriaxone, cefotaxime, imipenem and ceftazidime were prepared by dissolving in appropriate quantity of sterile distilled water.Varying proportion of Gentamicin and the other antibiotics ranging from 0:10 to 10:0 were mixed according to the continuous variation checkerboard method (6).Each proportion of the antibiotics in combination was serially diluted (2 fold) with sterile water.Th ereafter, 1 ml of each of the drug combinations was seeded in a Petri dish together with 19 ml of sterile nutrient agar.It was allowed to stand for 1 hour to solidify.An aliquot, 0.1ml equivalent of 0.5 ml MacFarland standard of ESBL producing organisms was streaked on the surface of the Mueller Hinton agar plate.Th e set up was done in triplicate with a control containing no antibiotics and these were incubated at 37oC for 24 hours and observed for growth.Th e MICs of the various combinations were determined and interactions between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration

RESULTS
Table 1 shows the result of the antimicrobial susceptibility study and minimum inhibitory concentration (MIC) (μg/ml).Th e MIC values recorded show rather staggered eff ects however the Klebsiella organisms displayed its values for the conventional cephalosporins and fl uroquinolones antibiotics.
Interestingly, this bacteria genus is associated with tremendous secretion and release of the beta-lactamase enzymes further correlating the thrust of this present research since they were selected (as we earlier reported in Afunwa et al, 2011) for further antibiotics combinatorial studies.We then quickly shifted to develop a simple antibiotics cocktail arrangement to be compatible with global settings including developing countries where cost of antibiotics treatment are crucial considerations towards good and eff ective clinical outcomes.Figures 1-5 present the outcome of the antibiotic combinatorial checkerboard assay.Gentamicin-pefl oxacin combinations (Figure 1) were largely synergistic against the community isolates in contrast to hospital isolates except Hp 3 and 4. It is a somewhat similar scenario for Gentamicin-Amoxicillin/Clavulanic acid cocktail (Figure 2) where again the community isolates were more susceptible synergistically than the hospital isolates.Th us, the same trend obtains for the Gentamicin-Amoxicillin/Clavulanic acid case.In the case of Gentamicin-Ceftriaxone (Figure 3), their combined eff ect holds no potential advantage among hospital isolates except in strain Hp4 where synergism is recorded.Th ere is also no remarkable utility against community strains where combined eff ect simply approached additivity.In a remarkable trend shift, in Figure 4, Gentamicin-Cefuroxime combination was clearly synergistic against community isolates.Nevertheless, hospitals isolates responded diff erently except isolate Hp4.Th e most potent synergism was recorded by Gentamicin-Ciprofl oxacin admix (Figure 5) against the plethora of

DISCUSSION
Th e wide use of antibiotics in the treatment of bacterial infections has led to the emergence and spread of resistant strains.Combined antibiotic therapy may produce synergistic eff ects in the treatment of bacterial infection and has been shown to delay the emergence of antimicrobial resistance (7-8).Antibi-otic combinations represent a therapeutic option in the treatment of a host of bacterial infections, as a result of the increasing appearance of multi-resistant microorganisms.In treatments involving antibiotics like rifampicin, combination therapy is used to avoid the appearance of antimicrobial resistance in the infectious agent (9)(10).In other treatments, combinations are used in order to enhance the eff ect of individual antimicrobials by means of synergic interactions.Th e optimal antibiotic combinations are commonly obtained from classical suscepti-

FIGURE 1. Susceptibility of ESBL Isolates to Gentamicinpefl oxacin antibiotics cocktail
Varying proportions of Gentamicin and Pefl oxacin antibiotics ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller Hinton agar.The ESBL producing organism was streaked on the surface of the agar plate.The set up was done in triplicate with a control containing no antibiotics and these were incubated at 37 o C for 24 hours and observed for growth.The MICs of the various combinations were determined and interactions between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration [FIC] index ratios where FIC index value below unity signifi es synergism, ≥1 ; indifference, and ≥2; antagonism (Cm1, Cm2, Cm5, and Cm6 represent community isolates, while Hp3, Hp4, Hp7, and Hp8 represent hospital isolates respectively).

FIGURE 2. Susceptibility of ESBL Isolates to Gentamicin-Amoxicillin/Clavulanic acid antibiotics cocktail
Varying proportions of Gentamicin and Amoxicillin/Clavulanic acid antibiotics ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller Hinton agar.The ESBL producing organism was streaked on the surface of the agar plate.The set up was done in triplicate with a control containing no antibiotics and these were incubated at 37 o C for 24 hours and observed for growth.The MICs of the various combinations were determined and interactions between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration [FIC] index ratios where FIC index value below unity signifi es synergism, ≥1 ; indifference, and ≥2; antagonism (Cm1, Cm2, Cm5, and Cm6 represent community isolates, while Hp3, Hp4, Hp7, and Hp8 represent hospital isolates respectively).
bility tests based on diff usion and dilution (broth microdilution test or Checkerboard), as described by the NCCLS.So far, the emergence of ESBL has compromised the utility of some currently preferred antibiotics of choice for some bacteria infectious diseases.Unfortunately, the low turnover rate for newer antibiotics to replace the compromised ones continues to hamper treatment.Th erefore, the possibility of re-inventing eff ective use protocol through fresh, seldom applied antibiotics combinatorial platform may prove a useful alternative approach to overcome this clinical fi sticuff s.Th ere are three potential advantages to using combination therapy: fi rstly, an increased likelihood that the infecting pathogen will be susceptible to at least one of the components of the regimen; secondly, prevention of emergence of resistance (11)(12) and thirdly, reduced mortality, perhaps because of an additive or even synergistic eff ect of the combination (13)(14)(15)(16).Checkerboard evaluations as a means of monitoring the combined activities of antimicrobial agents is based on the general outcome that FIC index value Varying proportions of Gentamicin and Ceftriaxone antibiotics ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller Hinton agar.The ESBL producing organism was streaked on the surface of the agar plate.The set up was done in triplicate with a control containing no antibiotics and these were incubated at 37 o C for 24 hours and observed for growth.The MICs of the various combinations were determined and interactions between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration [FIC] index ratios where FIC index value below unity signifi es synergism, ≥1 ; indifference, and ≥2; antagonism (Cm1, Cm2, Cm5, and Cm6 represent community isolates, while Hp3, Hp4, Hp7, and Hp8 represent hospital isolates respectively).

FIGURE 4. Susceptibility of ESBL Isolates to Gentamicin-Cefuroxime antibiotics cocktail
Varying proportions of Gentamicin and Cefuroxime antibiotics ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller Hinton agar.The ESBL producing organism was streaked on the surface of the agar plate.The set up was done in triplicate with a control containing no antibiotics and these were incubated at 37 o C for 24 hours and observed for growth.The MICs of the various combinations were determined and interactions between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration [FIC] index ratios where FIC index value below unity signifi es synergism, ≥1 ; indifference, and ≥2; antagonism (Cm1, Cm2, Cm5, and Cm6 represent community isolates, while Hp3, Hp4, Hp7, and Hp8 represent hospital isolates respectively).
below unity signifi es synergism, ≥1 ; indiff erence, and ≥2; antagonism.Th erefore, using checkerboard evaluation technique our investigation revealed that gentamicin-pefl oxacin combinations were largely synergistic against the hospital isolates in contrast to community isolates except Cm 3 and 4. It is however a somewhat opposite scenario for Gentamicin-Amoxicillin/Clavulanic acid cocktail where the community isolates were more susceptible synergistically than the hospital isolates.Th ese observations appear to have relationship to the use practice of the antibiotics in the settings.Gentamicin and pefl oxacin are usually largely abused among the populace within the communities (17) and as such could readily develop resistance against community bacteria isolates while maintaining eff ectiveness against the hospitals bacteria strains.Th e reverse trend appears true for the Gentamicin-Amoxicillin/Clavulanic acid case scenario.In the case of Gentamicin-Ceftriaxone, their combined eff ect holds no potential advantage among hospital isolates except in strain Cm4 where synergism is recorded.Th ere is also no remarkable utility against community strains where combined eff ect simply approached additivity.In a remarkable trend shift, Gentamicin-Cefuroxime combination was clearly synergistic against community isolates.Nevertheless, hospitals isolates responded diff erently except isolate Hp4.Th e most potent synergism was recorded by Gentamicin-Ciprofl oxacin admix against the plethora of the community strains.Th is was notably also reproduced in the strains Hp 3 and 4 of the hospital isolates, thus making this combination a most favourable cocktail for use against these ESBL-producing bacteria.Developing new therapeutic strategies using existing seldom utilized antimicrobial combinations still present useful option for tackling resistant microbial circumstances including the menace of the ESBLproducing bacteria (18)(19)(20).By simply adjusting the combinations and amounts of antibiotics applied could well be the fulcrum to tilt the balance in favour of eff ective therapy against these categories of infectious agents.Good antimicrobial monitoring exercise and corresponding antimicrobial screening activities should work towards a dynamic approach to generate eff ective treatment options using combination therapy.Th is would then fi ll a gaping void created by the ever-widening scourge of persistent and newer emerging resistant bacteria infections.

CONCLUSION
Th is particular study has shown the importance of keeping the wheel of progress in a dynamic state for continued antimicrobial solutions as cheap alternatives expected to fi nd practical usefulness in clinical settings especially in the tropics.Varying proportions of Gentamicin and Ciprofl oxacin antibiotics ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller Hinton agar.The ESBL producing organism was streaked on the surface of the agar plate.The set up was done in triplicate with a control containing no antibiotics and these were incubated at 37 o C for 24 hours and observed for growth.The MICs of the various combinations were determined and interactions between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration [FIC] index ratios where FIC index value below unity signifi es synergism, ≥1 ; indifference, and ≥2; antagonism (Cm1, Cm2, Cm5, and Cm6 represent community isolates, while Hp3, Hp4, Hp7, and Hp8 represent hospital isolates respectively).
[FIC] index using the relationship: FIC index = FIC A + FIC B 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 Th e activity index (AI) =log FIC index A and B are two antibiotics being combined; MIC, Minimum Inhibitory Concentration; Drug is antimicrobial agent.

FIGURE 3 .
FIGURE 3. Susceptibility of ESBL Isolates to Gentamicin-Ceftriaxone antibiotics cocktailVarying proportions of Gentamicin and Ceftriaxone antibiotics ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller Hinton agar.The ESBL producing organism was streaked on the surface of the agar plate.The set up was done in triplicate with a control containing no antibiotics and these were incubated at 37 o C for 24 hours and observed for growth.The MICs of the various combinations were determined and interactions between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration [FIC] index ratios where FIC index value below unity signifi es synergism, ≥1 ; indifference, and ≥2; antagonism (Cm1, Cm2, Cm5, and Cm6 represent community isolates, while Hp3, Hp4, Hp7, and Hp8 represent hospital isolates respectively).

FIGURE 5 .
FIGURE 5. Susceptibility of ESBL Isolates to Gentamicin-Ciprofl oxacin antibiotics cocktailVarying proportions of Gentamicin and Ciprofl oxacin antibiotics ranging from 1:9 to 9:1 were mixed according to the continuous variation checkerboard method and seeded into Mueller Hinton agar.The ESBL producing organism was streaked on the surface of the agar plate.The set up was done in triplicate with a control containing no antibiotics and these were incubated at 37 o C for 24 hours and observed for growth.The MICs of the various combinations were determined and interactions between the antimicrobial agents were assessed by determining their Fractional Inhibitory Concentration [FIC] index ratios where FIC index value below unity signifi es synergism, ≥1 ; indifference, and ≥2; antagonism (Cm1, Cm2, Cm5, and Cm6 represent community isolates, while Hp3, Hp4, Hp7, and Hp8 represent hospital isolates respectively).