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The synergistic effect of gentamicin and ceftazidime against Pseudomonas fluorescens

The synergistic effect of gentamicin and ceftazidime against Pseudomonas fluorescens BioscienceHorizons Volume 7 2014 10.1093/biohorizons/hzu007 Research article The synergistic effect of gentamicin and ceftazidime against Pseudomonas fluorescens Amy E. Morgan* Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, England *Corresponding author: Email: 1022579@chester.ac.uk Project Supervisor: Dr Neil Pickles, Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, England Email: n.pickles@chester.ac.uk. With antibiotic resistance becoming a significant problem in recent years, methodologies to overcome resistance have quickly become a necessity. One such mechanism to overcome resistance is to use antibiotics in combination. Clinical advice recom- mends the use of gentamicin and ceftazidime in combination to treat severe Pseudomonas aeruginosa infections. However, there is little evidence to support this recommendation. This study proposed that this recommendation is due to a synergistic effect and aimed to determine the optimum combination treatment, using Pseudomonas fluorescens as a model organism. Potentially, this research could give reason to a medical recommendation and even instigate a change in this treatment strat- egy in a clinical setting. To find the minimal inhibitory concentration (MIC) of the antibiotics used singularly, varying concen - trations of each antibiotic and P. fluorescens were placed in each well of a microtitre plate and incubated at 30°C for 24 h. Wells determined to have no growth were re-plated on nutrient agar and incubated at 30°C for 22 h for minimal bactericidal concen- tration (MBC) testing. When testing gentamicin and ceftazidime in combination, the checkerboard method was employed along with the fractional inhibitory concentration index (FICI) to test for synergy. A value of ≤0.5 defined synergy; 0.5 < FICI < 4 defined no interaction; ≥4 defined antagonism. No results of synergy were found; there were five results of no interaction and six results of antagonism. The MIC of ceftazidime was 3 µ g/ml and the MBC was 4 µ g/ml. The MIC of gentamicin was 0.25 µ g/ ml and the MBC was 3 µ g/ml. The combination of gentamicin and ceftazidime is optimal at a volume ratio of 1:1, in this case 25 µ l gentamicin/25 µ l ceftazidime, where gentamicin has a concentration of 0.5 µ g/ml and ceftazidime has a concentration of 0.25 µ g/ml, when used against 50 µ l of 1–2 × 10 colony forming units per millilitre of P. fluorescens in vitro. This study rec- ommends that this combination therapy be studied in depth in vivo, and that clinicians understand that this combination of antibiotics does not have a synergistic effect when treating patients in this manner. Key words: gentamicin, ceftazidime, Pseudomonas, combination therapy, synergy Submitted on 6 May 2014; accepted on 14 August 2014 Introduction and Vendan, 2007), leading to belief that human physiologi- cal temperature is not a barrier for the microorganism. There Pseudomonas fluorescens is a common environmental bacte- is evidence that a biofilm of P. fluorescens can be formed at rium, found in soil, in water and on plant surfaces (Guttman, 37°C (Donnarumma et al., 2010), and that glial cells exposed Morgan and Wang, 2008). Although considered non-patho- to P. fluorescens react with marked changes in nucleus mor- genic to humans, in recent years several clinical strains of P. phology, displaying typical changes of apoptotic mechanism fluorescens have been found to be able to survive at 37°C (Picot et al., 2001). This, along with recent clinical observa- (Chapalain et al., 2007), despite being considered as psychro- tions that P. fluorescens is a causative agent of nosocomial phile, with an optimum temperature of 25–30°C (Balachander infections, illustrates that some strains of P. fluorescens could © The Author 2014. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 7 2014 behave as a pathogen (Picot et al., 2001). There are several cases in which this bacterium has been found to be a human pathogen; however, in these cases, owing to the opportunistic nature of the bacterium, underlying conditions were present, for example, pathogenesis occurred in oncology and coro- nary care patients, as described by Benito et al. (2012), Gershman et al. (2008) and Hseuh et al. (1998). In these three cases, all patients recovered, some with the aid of anti- biotic treatment, and Benito et al. (2012) demonstrate that the bacterium is susceptible to both gentamicin and ceftazi- dime. Furthermore, 54% of individuals with Crohn’s disease were found to have a highly specific antigen of P. fluorescens (I2) in their serum, with a correlation of the severity of their condition and the level of circulating I2, illustrating an involvement in a multi-factorial disease (Madi et al., 2010). Although this study examines P. fluorescens , due to local- ized restrictions, this research could be extrapolated for use on the closely related, and more established pathogen, Pseudomonas aeruginosa (Dempsy, 2004). Although P. aeru- ginosa separated from other Pseudomonas species earlier than others, which allowed it to acquire a range of functions Figure 1. The chemical structure of (A) ceftazidime and (B) gentamicin. for it to become a dangerous opportunistic pathogen, P. fluo - (Reproduced with permission from Sigma-Aldrich Co . LLC (Sigma- Aldrich, 2012, 2013)). rescens is a relatively close relative, being closer than both Pseudomonas stutzeri, and Pseudomonas syringae in terms of genetic differences (Guttman, Morgan and Wang, 2008). There is minimal research as to why ceftazidime and gentami- cin should be used in conjunction when treating a P. aerugi- Pseudomonas aeruginosa, although rare as a community- nosa infection; however, it is recommended in hospital acquired pathogen (Huang, Lin and Wang, 2002), is among settings. This study is designed to give reason for this recom- the most common pathogens involved in nosocomial infec- mendation. One meta-analysis of published studies explained tions (Giamarellou, 2002), being the most common organism that their analysis of P. aeruginosa bacteraemia showed sig- (29%) to be isolated from postoperative wounds (Ranjan nificant benefits for patients using antibiotics in combination et al., 2011), with its presence also being found in various compared with patients receiving a single antibiotic, when other bodily sites (Aloush et al., 2006). Its presence in various looking at mortality rates. They found that there were more hospital sites such as hospital sinks, suction apparatus, air than double the amount deaths for patients receiving a single conditioning filters, operating tables and even on staff hands antibiotic, than patients receiving a combination of antibiot- could play a role in its prevalence (Pal, Rodrigues, and Datta, ics. However, this was only true for P. aeruginosa infections. 2010), leading to the bacteria often being named in the media Furthermore, of the 17 studies meeting the inclusion criteria, for its role in the infection of immunocompromised patients not one used ceftazidime and gentamicin as their combination (Hota et al., 2009) and premature neonates (Badr et al., 2011; of antibiotics (Safdar, Handelsman and Makim, 2004). BBC News Brisol, 2012; BBC News Northern Ireland, 2012). When presented with a patient suffering a P. aeruginosa infec- Only a few studies could be found that had used ceftazidime tion, it is recommended to use the third-generation cephalo- and gentamicin in combination, in vitro against P. aeruginosa. sporin ceftazidime with an aminoglycoside such as amikacin, Sputum samples from cystic fibrosis patients with chronic pul - gentamicin or tobramcyin as treatment (American Society of monary colonization of P. aeruginosa underwent direct sputum Health-System Pharmacists, 2004). Ceftazidime hydrate sensitivity testing using the Epsilometer test (Etest) method for (Fig. 1A) works by disrupting the synthesis of the peptidogly- MIC determination. The study revealed that 44% of samples can layer of the bacterial wall (Babic, Hujer and Bonomo, were susceptible to gentamicin and 53% of samples were sus- 2006) and has a broad spectrum of activity against both ceptible to ceftazidime. When looking at combination therapy, Gram-positive and Gram-negative bacteria, including both 21% of gentamicin/ceftazidime combinations showed synergy, P. fluorescens and P. aeruginosa (Richards and Brogden, 1985; and in 20% of cases, this was the most efficient combination Rains, Bryson and Peters, 1995). Likewise, gentamicin also (Serisier et al., 2012). Pruekprasert and Tunyapanit (2005) has a broad spectrum of activity, but it works by inhibiting found that when testing 50 P. aeruginosa isolates with ceftazi- bacterial protein synthesis by binding to the 30S subunit of dime and gentamicin in combination, 38.9% of the isolates the ribosome (Liou et al., 2006). In contrast to ceftazidime, showed synergy, defined as a fractional inhibitory concentra - however, gentamicin is made up of three major components: tion index (FICI) ≤0.5. They also found that in 44.4% of iso- C (C H N O ), C (C H N O ) and C (C H N O ); lates, an additive effect was observed (0.5 < FICI < 1.0), 16.7% 1 21 43 5 7 1a 20 41 5 7 2 19 39 5 7 usually <45% is C , <35% is C and <30% is C (Fig. 1B). of isolates exhibited an indifference effect (1.0 < FICI < 2.0), 1 1a 2 2 Bioscience Horizons • Volume 7 2014 Research article and that no isolates showed an antagonistic effect (FICI > 2). Test for synergy Shibi, Tawfik and Ramadan (1997) examined the effect of a Antibiotic combinations were evaluated by using the check- time interval between the administration of ceftazidime and erboard method. Gentamicin was diluted across the gamma- gentamicin in vitro to treat Pseudomonas aeruginosa and found irradiated 96-well U-bottom microtitre plate, in the wells, that adding the antibiotics at the same time had the best effect creating concentrations of 4, 2, 1, 0.5, 0.25, 0.125 and 0 µ g/ of lowering colony forming units (CFUs). ml. Dilutions of ceftazidime were premade in sterile universal Most clinicians agree that it is better to treat endocarditis bottles with concentrations of 4, 2, 1, 0.5, 0.25, 0.125 and and meningitis, caused by P. aeruginosa, with a bactericidal 0 µ g/ml, both using Mueller Hilton broth as a diluent. A total antibiotic (Seth and Seth, 2009); however, it does not matter of five microtitre plates were created with varying volumes of whether a bactericidal or bacteriostatic antibiotic is used for the two antibiotics, all with a total of 50 µ l. Plate 1 contained pneumonia or a urinary tract infection (Borton et al., 2011). 10 µ l of gentamicin and 40 µ l of ceftazidime; plate 2 con- As a study that could be applied in vivo, both the MIC and tained 20 µ l of gentamicin and 30 µ l of ceftazidime; plate 3 MBC therefore need determining. The checkerboard method contained 25 µ l of gentamicin and 25 µ l of ceftazidime; plate only tests for the MIC, for this study the MBC was also 4 contained 30 µ l of gentamicin and 20 µ l of ceftazidime; and tested, by taking a sample from wells with no visible growth plate 5 contained 40 µ l of gentamicin and 10 µ l of ceftazi- and plating it up on nutrient agar to see if growth returns dime. Once the plates had the antibiotics combinations in (Igbinosa et al., 2012). position, 50 µ l of 1–2 × 10 CFU/ml of bacterial suspension was pipetted into each well. The plates had their lids replaced The aim of this study is to determine whether the recom- and sealed with tape before being incubated at 30°C for 24 h. mendation by clinicians to use gentamicin and ceftazidime in MBC testing was also completed, as previously described combination as a treatment strategy for a P. aeruginosa infec- (Igbinosa et al., 2012). tion is due to these two antibiotics creating a synergistic effect, as there is little research to suggest why there is this recommen- MIC results were analysed using a FICI. FICI was calcu- dation. It is reasonable to hypothesize that this recommenda- lated as: tion is due to a synergistic effect. A second aim is to determine MICantibioticA MICantibiotiic B what combination of the two antibiotics is most effective. in combination in combination + . MICantibioticA alone MICantibioticB alone Methods A value of ≤0.5 defined synergy; a value of 0.5 < FICI < 4 MIC and MBC tests defined no interaction; a value of ≥4 defined antagonism (Mitsugui et al., 2011). Using aseptic techniques throughout, the bacterial strain P. fluorescens NCIB 9046 was suspended in 0.85% saline Statistical tests solution (until equal to 0.5 McFarland solution). Then 1 ml of this solution was placed into 99 ml of Mueller Hilton broth, The statistical tests were selected after determining that the creating a bacterial suspension of 1–2 × 10 CFU/ml (Choi data were normally distributed (parametric), using SPSS ver- and Lee, 2012; Igbinosa et al., 2012). To find the MIC of each sion 18. A p value <0.05 was considered statistically signifi - antibiotic, 50 µ l of 256–0 µ g/ml of ceftazidime (Ceftazidime cant. Using the data from the antibiotics used singularly, the Hydrate, Sigma life science, C3809–1G, lot: SLBD0920V) independent samples Mann–Whitney U test was employed to and gentamicin (Gentamicin injectable, for IM or IV injection, test that the two antibiotics had the same concentration dis- 80 mg in 2 ml, Gentacin, 051365) was placed in triplicate tribution. Further using this data, both the Pearson’s correla- wells of a gamma-irradiated 96-well U-bottom microtitre tion and Spearman’s rho analyses were used to test whether plate (separate plate for each antibiotic). Dilutions were cre- there was a correlation between the amount of growth visi- ated in the wells using Mueller Hilton broth as a diluent. ble, after the initial incubation, and the strength of the anti- To this, 50 µ l of 1–2 × 10 CFU/ml of the aforementioned bac- biotics. To test whether there was a difference in the MIC terial suspension was added to each well, and each plate had values of antibiotics used singularly, a Chi-squared test was the lid replaced and sealed with tape before being placed in used. To test whether there was a difference in the amount of the incubator at 30°C for 24 h. The MIC was defined as the growth, after the initial incubation, between the five combi - lowest dilution of antibiotic which completely inhibited the nation plates, a chi-squared test was employed. growth of P. fluorescens , with growth determined as a cream/ white dot in the centre of a well (Igbinosa et al., 2012). For Results MBC testing, a loop of solution from wells not containing growth after the initial incubation was spread onto nutrient MIC and MBC tests agar and incubated for a further 22 h at 30°C. The MBC was determined as the lowest dilution of antibiotic in which no For the MIC test for ceftazidime used against Pseudomonas growth was seen on the agar plate, where growth was deter- fluorescens , the MIC was determined as 3 µ g/ml, as it was the mined as cream/white colonies (Igbinosa et al., 2012). lowest concentration to show no growth. For the MBC test 3 Research article Bioscience Horizons • Volume 7 2014 for ceftazidime used against P. fluorescens , the concentra- occurred; thus, clinical recommendations to use ceftazidime tions within the range of 256–3 µ g/ml were plated on nutri- and gentamicin in combination to treat a Pseudomonas infec- ent agar in triplicate. All three triplicate wells containing tion are not due to a synergistic effect. 3 µ g/ml showed growth, and so the MBC was determined as Ratio of antibiotics in combination analysis 4 µ g/ml. For the MIC test for gentamicin used against P. fluo - rescens, the MIC was determined as 0.25 µ g/ml, as it was the There is a statistically significant difference in the amount of lowest concentration to show no growth. Although 1 out of growth between the five combinations of gentamicin and the 3 wells containing 0.75 µ g/ml of gentamicin had growth ceftazidime used, after the initial incubation, with a signifi - present, this was considered as an anomalous result and dis- cance of <0.01 when tested by a chi-squared test. Figure 3 regarded. For the MBC test for gentamicin used against illustrates that when looking at growth after the original P. fluorescens , concentrations 256–0.25 µ g/ml were plated on incubation, as the volume of gentamicin increases and the nutrient agar in triplicate. All three triplicate wells containing ceftazidime decreases, the number of wells showing growth 0.25–2 µ g/ml gentamicin showed growth, and so the MBC decreases. This shows that combinations of 30 µ l gentami- was determined as 3 µ g/ml (Table 1). cin/20 µ l ceftazidime and 40 µ l gentamicin/10 µ l ceftazidime have the greatest bacteriostatic effect against P. fluorescens as As the concentration of both antibiotics increases, when only 21 wells (43%) showed growth in comparison to 35 used singularly, the amount of growth seen decreases; this wells (71%) growth for the combination 10 µ l gentami- illustrates that there is a negative correlation between the cin/40 µ l ceftazidime. The combination 25 µ l gentami- concentration of both antibiotics used singularly and the cin/25 µ l ceftazidime had the least amount of growth return, amount of growth, significant to the <0.01 level, when statis- only one well (2%), when re-plated for the MBC test, illus- tically tested by both the Pearson correlation and Spearman’s trating that this combination had the best bactericidal effect rho. Furthermore, there is a statistically significant difference on P. fluorescens . A combination of 30 µ l gentamicin/20 µ l in the MICs of gentamicin and ceftazidime when used singu- ceftazidime and 40 µ l gentamicin/10 µ l ceftazidime had the larly, when statistically tested with the chi-squared test, with most amount of re-growth when re-plated for the MBC test a significance of <0.01. (20 and 22%, respectively). When looking at the total FICI values for antibiotics in combination amount of growth, growth after the original incubation and further incubation on nutrient agar, the combination of 25 µ l A value of ≤0.5 illustrates synergy; 0.5 < FICI < 4 illustrates no gentamicin/25 µ l ceftazidime has the fewest number of wells, interaction between antibiotics; and ≥4 illustrates antagonism. only 25, showing growth (51%) in comparison to 39 wells Interpreted values will demonstrate whether a synergistic effect (79%) for the 10 µ l gentamicin/40 µ l ceftazidime combina- occurs, giving reason to clinical recommendations to use these tion. The optimal combination of antibiotics comes from the two antibiotics in combination. Plate 1: The FICI values 8.17 25 µ l gentamicin/25 µ l ceftazidime plate, from well F5 where and 4.3 indicate that the two antibiotics are antagonizing each gentamicin has a concentration of 0.5 µ g/ml and ceftazidime other’s antimicrobial activity and hence raising their MIC val- has a concentration of 0.25 µ g/ml, when used against ues. Plate 2: When looking at the FICI values, both 8.04 and 1–2 × 10 CFU/ml of P. fluorescens . 5.3 indicate that the two antibiotics are antagonizing each other’s antimicrobial activity and hence raising their MIC val- Discussion ues. Plate 3: When looking at the calculated FICI value of 2.04, it is illustrated that there is no interaction between the two Pseudomonas fluorescens was susceptible to both gentamicin antibiotics, in that the MIC values remain unaltered by the and ceftazidime as Benito et al. (2012) also found. Extensive other, neither positively nor negatively. Plates 4 and 5: These searches for relevant research regarding these compounds two plates have matching FICI calculations. The well G4 has a and the effect of synergy have yielded little published research calculated FICI value of 4.04 which indicated that the two for P. fluorescens. These results can however be extrapolated antibiotics are antagonizing one another’s antimicrobial activ- to Pseudomonas aeruginosa as P. fluorescens is one of its ity and hence raising the MIC values. However, wells F5 and closest relatives and, in addition, Richards and Brogden E6 have produced FICI values of 2.83 and 1.17, respectively, (1985) and Mikura et al. (2011) state that gentamicin and and this indicates that the antibiotics are neither positively nor ceftazidime are also effective against the more established negatively interacting with one another, to alter the MIC values human pathogen, P. aeruginosa. of one another (Fig. 2 and Table 2). No results of synergy The MIC of ceftazidime was determined to be 3 µ g/ml and Table 1. MIC and MBC results for gentamicin and ceftazidime the MBC to be 4 µ g/ml against P. fluorescen s. This MIC value is within the range of 2–128 µ g/ml as stated by Tunney and Scott (2004) and within the range of 0.5–128 µ g/ml for Antibiotic MIC (µ g/ml) MBC (µ g/ml) P. aeruginosa, stated by Richards and Brogden (1985) and Gentamicin 0.25 3 Rains, Bryson and Peters (1995), who also state that the MBC values are either the same or less than twice the MIC, Ceftazidime 3 4 which the results in this study support. 4 Bioscience Horizons • Volume 7 2014 Research article Figure 2. Pseudomonas fluorescens growth when gentamicin and ceftazidime are used in combination. (A) A volume of 10 µ l of gentamicin and 40 µ l ceftazidime for concentrations ranging from 4 to 0 µ g/ml are in each well-concentrations only shown on (A) but are the same throughout the plates. (B) A volume of 20 µ l of gentamicin and a 30 µ l of ceftazidime of concentrations ranging from 4 to 0 µ g/ml are in each well. (C) A volume of 25 µ l of gentamicin and a 25 µ l of ceftazidime of concentrations ranging from 4 to 0 µ g/ml are in each well. (D) A volume of 30 µ l of gentamicin and a 20 µ l of ceftazidime of concentrations ranging from 4 to 0 µ g/ml are in each well. (E) A volume of 40 µ l of gentamicin and a 10 µ l of ceftazidime of concentrations ranging from 4 to 0 µ g/ml are in each well. To each well, 50 µ l of 1–2 × 10 CFU/ml of P. fluorescens suspension is added. Filled circles represent wells where growth occurred after incubation at 30°C for 24 h. Open circles represent wells where no growth occurred after incubation at 30°C for 24 h. Growth was recognized as a white/cream dot in the centre of the well (MIC test). Wells showing no growth were plated onto nutrient agar for the MBC test. They were incubated at 30°C for 22 h. Circles with cross lines represents re-growth on nutrient agar, recognized as visible cream colonies on the agar (n = 1). The MIC of gentamicin was determined to be 0.25 µ g/ml Gyselynck, Forrey and Cutler (1971) illustrated the ototoxic and the MBC to be 3 µ g/ml against P. fluorescen s. This is effects when high levels of gentamicin are used in vivo. lower than the range of 1–16 µ g/ml for P. aeruginosa stated However, Richards and Brogden (1985) and Rains, Bryson by Burgess and Nathisuwan (2002). The MBC is also lower and Peters (1995) discussed that ceftazidime is eliminated than >8 µ g/ml stated by Rukholm et al. (2006). This could quickly from the body via the urine, and for this reason, it has mean that P. fluorescens is more susceptible to gentamicin limited side effects. Due to the side effects of gentamicin and than P. aeruginosa. However, Sheppard (unpublished data) the quick elimination time of ceftazidime, it would be sensible found the same MIC value as this study, when using the same to believe that using a combination in which the gentamicin stock of gentamicin and same strain of P. fluorescens . level is lowest would be more desirable, for use in vivo, when looking at all of the combinations that produce a desired effect. Safdar, Handelsman and Makim (2004) explain that from conducting a meta-analysis, the use of a combination of anti- Seth and Seth (2009) describe that it is better to treat biotics is more beneficial on mortality rates than using an severe P. aeruginosa infections with a bactericidal antibiotic, antibiotic singularly. During this study, no results of synergy which illustrates that the choice of the 25 µ l gentamicin/25 µ l were produced (FICI ≤ 0.5); however, results of no interac- ceftazidime plate is the best choice, as the lowest amount of tion (0.5 < FICI < 4) were produced. Considering that it is re-growth occurred from this plate, and has a lower volume beneficial to use a combination therapy, the use of the ‘no of gentamicin compared with 30 µ l gentamicin/20 µ l ceftazi- interaction’ results could be used in vivo, with perhaps a ben- dime and 40 µ l gentamicin/10 µ l ceftazidime. eficial effect on the patient, and this study has determined the However, when considering that it is better to treat severe best combination to use. P. aeruginosa infections with a bactericidal antibiotic it would Balakumar, Rohilla and Thangathirupathi (2010) illus- perhaps be beneficial to use combination of antibiotics found in trated the nephrotoxic effects, and Zaske et al. (1981) and well F5 rather than in G5. Well F5 (25 µ l of 0.5 µg/ml gentamicin 5 Research article Bioscience Horizons • Volume 7 2014 and 25 µ l of 0.25 µ g/ml ceftazidime) would produce a FICI of ratio of 1:1, in this case 25 µ l gentamicin/25 µ l ceftazidime, 2.83, which would still demonstrate no interaction between where gentamicin has a concentration of 0.5 µ g/ml and ceftazi- antibiotics. This study shows that it may be best to suggest well dime has a concentration of 0.25 µ g/ml, when used against F5 is more appropriate, because re-growth does not occur at this 1–2 × 10 CFU/ml of P. fluorescens . level, and it is the lowest concentration of gentamicin possible. Antibiotic resistance has become more prevalent in recent Considering this, this study demonstrates that the most practical years, leading to methodologies for treating infection without combination of gentamicin and ceftazidime is to use a volume the concern of antibiotic resistance being needed more than ever. One such method for overcoming antibiotic resistance is Table 2. FICI calculation and interpretation from the checkerboard to use existing antibiotics in combination. The aim of the method results study was to determine whether clinical recommendations to use ceftazidime and gentamicin in combination against Plate Well Calculation Result Interpretation P. aeruginosa are due to a synergistic effect. This study deter- mines that a synergistic effect does not occur, and that this 1 E3 2/0.25 + 0.5/3 8.17 Antagonism recommendation is due to evidence that combination therapy increases a patient’s likelihood of survival compared with a D4 1/0.25 + 1/3 4.3 Antagonism single antibiotic treatment. Although synergy was not found 2 G4 2/0.25 + 0.125/3 8.04 Antagonism in this study, it may prove beneficial to use this combination in vivo. The major limitation of this study is the lack of rep- B5 1/0.25 + 4/3 5.3 Antagonism lication, and this study recommends this treatment be repli- cated in vitro and studied in depth in vivo. 3 G5 0.5/0.25 + 0.125/3 2.04 No interaction 4 G4 1/0.25 + 0.125/3 4.04 Antagonism Author biography F5 0.5/0.25 + 0.25/3 2.83 No interaction A.E.M. has just graduated with a First in BSc Biomedical E6 0.25/0.25 + 0.5/3 1.17 No interaction Sciences from the University of Chester, where during her studies here she gained a passion for research, especially 5 G4 1/0.25 + 0.125/3 4.04 Antagonism within the field of microbiology. For this reason, she is cur - rently studying for a Master of Research (MRes) in Applied F5 0.5/0.25 + 0.25/3 2.83 No interaction Science, at the same university, where her focus is on the E6 0.25/0.25 + 0.5/3 1.17 No interaction Quorum sensing and quorum quenching of Chromobacterium violaceum. Her hopes for the future are to go on to complete Furthest bottom right wells were selected for FICI analysis; in the case of plates 1, 2, 4 and 5, more than one well was chosen for analysis as a staggered a PhD and gain a career in research focusing on antibiotic result was created, as seen in Figure 2. resistance and alternative antimicrobial therapies. Figure 3. The total amount of growth, including original growth, and re-growth, for all five combinations of gentamicin and ceftazidime. G represents gentamicin, C represents ceftazidime. Each well contained a total of 50 µ l of antibiotic and 50 µ l of 1–2 × 10 CFU/ml of Pseudomonas fluorescens suspension. Filled boxes represent the number of wells that showed growth after the original incubation at 30°C for 24 h. Wells that showed no growth were plated onto nutrient agar for the MBC test. They were incubated at 30°C for 22 h. Boxes with lines represent the number of wells where re-growth occurred on nutrient agar (n = 1). 6 Bioscience Horizons • Volume 7 2014 Research article Dempsy, M. (2004) The Approved List of Biological Agents, Advisory Acknowledgements Committee on Dangerous Pathogens, Colegate. I would like to thank my supervisor Dr Neil Pickles for his Donnarumma, G., Buommino, E., Fusco, A. et  al. (2010) Effect of tem - expert advice and support throughout the research process, perature on the shift of Pseudomonas fluorescens from an environ- along with technical staff member Mary Wood for the guid- mental microorganism to a potential human pathogen, International ance she provided for the methodology. Journal of Immunopathology and Pharmacology, 23(1), 227–34. Gershman, M. D., Kennedy, D. J., Nobel-Wang, J. et al. (2008) Multistate Funding outbreak of Pseudomonas fluorescens bloodstream infection after exposure to contaminated heparinized saline flush prepared by a The Department of Biological Science, of the University of compounding pharmacy, Clinical Infectious Diseases, 47(11), Chester, funded this research. 1372–9. References Giamarellou, H. (2002) Prescribing guidelines for severe Pseudomonas infections, Journal of Antimicrobial Chemotherapy, 49(2), 229–33. Aloush, V., Navon-Venezia, S., Seigman-Igra, Y. et  al. (2006) Multidrug- resistant Pseudomonas aeruginosa: risk factors and clinical impact, Guttman, D. S., Morgan, R. L. and Wang, P. W. (2008) The evolution of the Antimicrobial Agents and Chemotherapy, 50(1), 43–8. Pseudomonads, In: M. Fatmi, A. Collmer, N. S. Iacobellis, J. W. Mansfield, J. Murillo, N. W. Schaad, and M. Ullrich (eds) Pseudomonas American Society of Health-System Pharmacists, I. 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(2012) Timeline: pseudomonas outbreak in ergy interaction against multidrug-resistant Pseudomonas aerugi- Londonderry and Belfast, BBC News, Northern Ireland, Belfast. nosa isolated from an abattoir effluent environment, The Scientific World Journal, 2012 (308034), 1–5. Benito, N., Mirelis, B., Galvez, M. L. et al. (2012) Outbreak of Pseudomonas fluorescens bloodstream infection in a coronary care unit, Journal of Liou, G. F., Yoshizawa, S., Courvalin, P. et al. (2006) Aminoglycoside resis- Hospital Infection, 82(4), 286–9. tance by ArmA-mediated ribosomal 16 S methylation in human bac- terial pathogens, Journal of Molecular Biology, 2(2), 358–64. Borton, D., Brinsko, V., Gilmore, G. K. et  al. (2011) Lippincott’s Guide to Infectious Diseases, Lipincott Williams & Wilkins, Norristown. Madi, A., Svinareff, P., Orange, N. et  al. (2010) Pseudomonas fluorescens Burgess, D. S. and Nathisuwan, S. (2002) Cefepime, piperacillin/tazobactam, alters epithelial permeability and translocates across Caco-2/TC7 gentamicin, ciprofloxacin, and levofloxacin alone and in combin ation intestinal cells, Gut Pathogens, 2(16), 1–8. against Pseudomonas aeruginosa, Diagnostic Microbiology and Mikura, S., Wada, H., Okazaki, M. et al. (2011) Risk factors for bacterae- Infectious Disease, 44(1), 35–41. mia attributable to Pseudomonas aeruginosa resistant to imipe- Chapalain, A., Rossignol, G., Lesouhaitier, O. et  al. (2007) Comparative nem, levofloxacin, or gentamicin, Journal of Hospital Infection, study of 7 fluorescent pseudomonad clinical isolates, Canadian 79(3), 267–8. Journal of Microbiology, 54(1), 19–27. Mitsugui, C. S., Bronharo-Tognim, M. C., Cardoso, C. L. et  al. (2011) In Choi, H. and Lee, D. G. 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(2009) Textbook of Pharmacology, 3rd edn., and Infection, 3(12), 985–95. Elsevier, New Delhi. Pruekprasert, P. and Tunyapanit, W. (2005) In vitro activity of fosfomycin- Sheppard, L. (Unpublished data) Investigating adaptive resistance to gentamicin, fosfomycin-ceftazidime, fosfomycin-imipenem and aminoglycosides, 1–36. ceftazidime-gentamicin combinations against ceftazidime-r esistant Shibi, A. M., Tawfik, A. F. and Ramadan, M. A. (1997) Comparative efficacy Pseudomonas aeruginosa, Southeast Asian Journal of Tropical of successive exposure of Pseudomonas aeruginosa to gentamicin Medicine and Public Health, 36(5), 1239–42. and ceftazidime. International Journal of Antimicrobial Agents, 8, Rains, C. P., Bryson, H. M. and Peters, D. H. (1995) Ceftazidime: an update 257–61. of its antibacterial activity, pharmacokinetic properties and thera- Sigma-Aldrich. (2012) Gentamicin solution. Retrieved from Sigma- peutic efficacy, Drugs, 49(4), 577–617. Aldrich, accessed at: http://www.sigmaaldrich.com/catalog/product/ Ranjan, K. P., Ranjan, N., Bansal, S. K. et  al. (2011) Prevalence of sigma/g1397?lang=en&region=GB (16 September 2014). Pseudomonas aeruginosa in post-operative wound infection in a Sigma-Aldrich. (2013) Ceftazidime hydrate. Retrieved from Sigma- referral hospital in Haryana, India, Journal of Laboratory Physicians, Aldrich, accessed at: http://www.sigmaaldrich.com/catalog/product/ 2(2), 129–32. sigma/c3809?lang=en&region=GB (16 September 2014). Richards, D. M. and Brogden, R. N. (1985) Ceftazidime: a review of its Tunney, M. M. and Scott, E. M. (2004) Use of breakpoint combination antibacterial activity, pharmacokinetic properties and therapeutic sensitivity testing as a simple and convenient method to evaluate use, Drugs, 29(2), 105–61. the combined effects of ceftazidime and tobramycin on Rukholm, G., Mugabe, C., Azghani, A. O. et al. (2006) Antibacterial activity Pseudomonas aeruginosa and Burkholderia cepacia complex isolates of liposomal gentamicin against Pseudomonas aeruginosa: a time– in vitro, Journal of Microbiological Methods, 57(1), 107–14. kill study, International Journal of Antimicrobial Agents, 27(3), 247–52. Zaske, D. E., Cipolle, R. J., Rotschafer, J. C. et  al. (1981) Gentamicin Safdar, N., Handelsman, J. and Makim, D. G. (2004) Does combination pharmacokinetics in 1,640 patients: method for control of serum antimicrobial therapy reduce mortality in Gram-negative bacterae- concentrations, Antimicrobial Agents and Chemotherapy, 21(3), mia? A meta-analysis, The Lancet, 4, 519–27. 407–11. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

The synergistic effect of gentamicin and ceftazidime against Pseudomonas fluorescens

Morgan, Amy E.
Bioscience Horizons , Volume 7 – Sep 25, 2014
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BioscienceHorizons Volume 7 2014 10.1093/biohorizons/hzu007 Research article The synergistic effect of gentamicin and ceftazidime against Pseudomonas fluorescens Amy E. Morgan* Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, England *Corresponding author: Email: 1022579@chester.ac.uk Project Supervisor: Dr Neil Pickles, Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, England Email: n.pickles@chester.ac.uk. With antibiotic resistance becoming a significant problem in recent years, methodologies to overcome resistance have quickly become a necessity. One such mechanism to overcome resistance is to use antibiotics in combination. Clinical advice recom- mends the use of gentamicin and ceftazidime in combination to treat severe Pseudomonas aeruginosa infections. However, there is little evidence to support this recommendation. This study proposed that this recommendation is due to a synergistic effect and aimed to determine the optimum combination treatment, using Pseudomonas fluorescens as a model organism. Potentially, this research could give reason to a medical recommendation and even instigate a change in this treatment strat- egy in a clinical setting. To find the minimal inhibitory concentration (MIC) of the antibiotics used singularly, varying concen - trations of each antibiotic and P. fluorescens were placed in each well of a microtitre plate and incubated at 30°C for 24 h. Wells determined to have no growth were re-plated on nutrient agar and incubated at 30°C for 22 h for minimal bactericidal concen- tration (MBC) testing. When testing gentamicin and ceftazidime in combination, the checkerboard method was employed along with the fractional inhibitory concentration index (FICI) to test for synergy. A value of ≤0.5 defined synergy; 0.5 < FICI < 4 defined no interaction; ≥4 defined antagonism. No results of synergy were found; there were five results of no interaction and six results of antagonism. The MIC of ceftazidime was 3 µ g/ml and the MBC was 4 µ g/ml. The MIC of gentamicin was 0.25 µ g/ ml and the MBC was 3 µ g/ml. The combination of gentamicin and ceftazidime is optimal at a volume ratio of 1:1, in this case 25 µ l gentamicin/25 µ l ceftazidime, where gentamicin has a concentration of 0.5 µ g/ml and ceftazidime has a concentration of 0.25 µ g/ml, when used against 50 µ l of 1–2 × 10 colony forming units per millilitre of P. fluorescens in vitro. This study rec- ommends that this combination therapy be studied in depth in vivo, and that clinicians understand that this combination of antibiotics does not have a synergistic effect when treating patients in this manner. Key words: gentamicin, ceftazidime, Pseudomonas, combination therapy, synergy Submitted on 6 May 2014; accepted on 14 August 2014 Introduction and Vendan, 2007), leading to belief that human physiologi- cal temperature is not a barrier for the microorganism. There Pseudomonas fluorescens is a common environmental bacte- is evidence that a biofilm of P. fluorescens can be formed at rium, found in soil, in water and on plant surfaces (Guttman, 37°C (Donnarumma et al., 2010), and that glial cells exposed Morgan and Wang, 2008). Although considered non-patho- to P. fluorescens react with marked changes in nucleus mor- genic to humans, in recent years several clinical strains of P. phology, displaying typical changes of apoptotic mechanism fluorescens have been found to be able to survive at 37°C (Picot et al., 2001). This, along with recent clinical observa- (Chapalain et al., 2007), despite being considered as psychro- tions that P. fluorescens is a causative agent of nosocomial phile, with an optimum temperature of 25–30°C (Balachander infections, illustrates that some strains of P. fluorescens could © The Author 2014. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 7 2014 behave as a pathogen (Picot et al., 2001). There are several cases in which this bacterium has been found to be a human pathogen; however, in these cases, owing to the opportunistic nature of the bacterium, underlying conditions were present, for example, pathogenesis occurred in oncology and coro- nary care patients, as described by Benito et al. (2012), Gershman et al. (2008) and Hseuh et al. (1998). In these three cases, all patients recovered, some with the aid of anti- biotic treatment, and Benito et al. (2012) demonstrate that the bacterium is susceptible to both gentamicin and ceftazi- dime. Furthermore, 54% of individuals with Crohn’s disease were found to have a highly specific antigen of P. fluorescens (I2) in their serum, with a correlation of the severity of their condition and the level of circulating I2, illustrating an involvement in a multi-factorial disease (Madi et al., 2010). Although this study examines P. fluorescens , due to local- ized restrictions, this research could be extrapolated for use on the closely related, and more established pathogen, Pseudomonas aeruginosa (Dempsy, 2004). Although P. aeru- ginosa separated from other Pseudomonas species earlier than others, which allowed it to acquire a range of functions Figure 1. The chemical structure of (A) ceftazidime and (B) gentamicin. for it to become a dangerous opportunistic pathogen, P. fluo - (Reproduced with permission from Sigma-Aldrich Co . LLC (Sigma- Aldrich, 2012, 2013)). rescens is a relatively close relative, being closer than both Pseudomonas stutzeri, and Pseudomonas syringae in terms of genetic differences (Guttman, Morgan and Wang, 2008). There is minimal research as to why ceftazidime and gentami- cin should be used in conjunction when treating a P. aerugi- Pseudomonas aeruginosa, although rare as a community- nosa infection; however, it is recommended in hospital acquired pathogen (Huang, Lin and Wang, 2002), is among settings. This study is designed to give reason for this recom- the most common pathogens involved in nosocomial infec- mendation. One meta-analysis of published studies explained tions (Giamarellou, 2002), being the most common organism that their analysis of P. aeruginosa bacteraemia showed sig- (29%) to be isolated from postoperative wounds (Ranjan nificant benefits for patients using antibiotics in combination et al., 2011), with its presence also being found in various compared with patients receiving a single antibiotic, when other bodily sites (Aloush et al., 2006). Its presence in various looking at mortality rates. They found that there were more hospital sites such as hospital sinks, suction apparatus, air than double the amount deaths for patients receiving a single conditioning filters, operating tables and even on staff hands antibiotic, than patients receiving a combination of antibiot- could play a role in its prevalence (Pal, Rodrigues, and Datta, ics. However, this was only true for P. aeruginosa infections. 2010), leading to the bacteria often being named in the media Furthermore, of the 17 studies meeting the inclusion criteria, for its role in the infection of immunocompromised patients not one used ceftazidime and gentamicin as their combination (Hota et al., 2009) and premature neonates (Badr et al., 2011; of antibiotics (Safdar, Handelsman and Makim, 2004). BBC News Brisol, 2012; BBC News Northern Ireland, 2012). When presented with a patient suffering a P. aeruginosa infec- Only a few studies could be found that had used ceftazidime tion, it is recommended to use the third-generation cephalo- and gentamicin in combination, in vitro against P. aeruginosa. sporin ceftazidime with an aminoglycoside such as amikacin, Sputum samples from cystic fibrosis patients with chronic pul - gentamicin or tobramcyin as treatment (American Society of monary colonization of P. aeruginosa underwent direct sputum Health-System Pharmacists, 2004). Ceftazidime hydrate sensitivity testing using the Epsilometer test (Etest) method for (Fig. 1A) works by disrupting the synthesis of the peptidogly- MIC determination. The study revealed that 44% of samples can layer of the bacterial wall (Babic, Hujer and Bonomo, were susceptible to gentamicin and 53% of samples were sus- 2006) and has a broad spectrum of activity against both ceptible to ceftazidime. When looking at combination therapy, Gram-positive and Gram-negative bacteria, including both 21% of gentamicin/ceftazidime combinations showed synergy, P. fluorescens and P. aeruginosa (Richards and Brogden, 1985; and in 20% of cases, this was the most efficient combination Rains, Bryson and Peters, 1995). Likewise, gentamicin also (Serisier et al., 2012). Pruekprasert and Tunyapanit (2005) has a broad spectrum of activity, but it works by inhibiting found that when testing 50 P. aeruginosa isolates with ceftazi- bacterial protein synthesis by binding to the 30S subunit of dime and gentamicin in combination, 38.9% of the isolates the ribosome (Liou et al., 2006). In contrast to ceftazidime, showed synergy, defined as a fractional inhibitory concentra - however, gentamicin is made up of three major components: tion index (FICI) ≤0.5. They also found that in 44.4% of iso- C (C H N O ), C (C H N O ) and C (C H N O ); lates, an additive effect was observed (0.5 < FICI < 1.0), 16.7% 1 21 43 5 7 1a 20 41 5 7 2 19 39 5 7 usually <45% is C , <35% is C and <30% is C (Fig. 1B). of isolates exhibited an indifference effect (1.0 < FICI < 2.0), 1 1a 2 2 Bioscience Horizons • Volume 7 2014 Research article and that no isolates showed an antagonistic effect (FICI > 2). Test for synergy Shibi, Tawfik and Ramadan (1997) examined the effect of a Antibiotic combinations were evaluated by using the check- time interval between the administration of ceftazidime and erboard method. Gentamicin was diluted across the gamma- gentamicin in vitro to treat Pseudomonas aeruginosa and found irradiated 96-well U-bottom microtitre plate, in the wells, that adding the antibiotics at the same time had the best effect creating concentrations of 4, 2, 1, 0.5, 0.25, 0.125 and 0 µ g/ of lowering colony forming units (CFUs). ml. Dilutions of ceftazidime were premade in sterile universal Most clinicians agree that it is better to treat endocarditis bottles with concentrations of 4, 2, 1, 0.5, 0.25, 0.125 and and meningitis, caused by P. aeruginosa, with a bactericidal 0 µ g/ml, both using Mueller Hilton broth as a diluent. A total antibiotic (Seth and Seth, 2009); however, it does not matter of five microtitre plates were created with varying volumes of whether a bactericidal or bacteriostatic antibiotic is used for the two antibiotics, all with a total of 50 µ l. Plate 1 contained pneumonia or a urinary tract infection (Borton et al., 2011). 10 µ l of gentamicin and 40 µ l of ceftazidime; plate 2 con- As a study that could be applied in vivo, both the MIC and tained 20 µ l of gentamicin and 30 µ l of ceftazidime; plate 3 MBC therefore need determining. The checkerboard method contained 25 µ l of gentamicin and 25 µ l of ceftazidime; plate only tests for the MIC, for this study the MBC was also 4 contained 30 µ l of gentamicin and 20 µ l of ceftazidime; and tested, by taking a sample from wells with no visible growth plate 5 contained 40 µ l of gentamicin and 10 µ l of ceftazi- and plating it up on nutrient agar to see if growth returns dime. Once the plates had the antibiotics combinations in (Igbinosa et al., 2012). position, 50 µ l of 1–2 × 10 CFU/ml of bacterial suspension was pipetted into each well. The plates had their lids replaced The aim of this study is to determine whether the recom- and sealed with tape before being incubated at 30°C for 24 h. mendation by clinicians to use gentamicin and ceftazidime in MBC testing was also completed, as previously described combination as a treatment strategy for a P. aeruginosa infec- (Igbinosa et al., 2012). tion is due to these two antibiotics creating a synergistic effect, as there is little research to suggest why there is this recommen- MIC results were analysed using a FICI. FICI was calcu- dation. It is reasonable to hypothesize that this recommenda- lated as: tion is due to a synergistic effect. A second aim is to determine MICantibioticA MICantibiotiic B what combination of the two antibiotics is most effective. in combination in combination + . MICantibioticA alone MICantibioticB alone Methods A value of ≤0.5 defined synergy; a value of 0.5 < FICI < 4 MIC and MBC tests defined no interaction; a value of ≥4 defined antagonism (Mitsugui et al., 2011). Using aseptic techniques throughout, the bacterial strain P. fluorescens NCIB 9046 was suspended in 0.85% saline Statistical tests solution (until equal to 0.5 McFarland solution). Then 1 ml of this solution was placed into 99 ml of Mueller Hilton broth, The statistical tests were selected after determining that the creating a bacterial suspension of 1–2 × 10 CFU/ml (Choi data were normally distributed (parametric), using SPSS ver- and Lee, 2012; Igbinosa et al., 2012). To find the MIC of each sion 18. A p value <0.05 was considered statistically signifi - antibiotic, 50 µ l of 256–0 µ g/ml of ceftazidime (Ceftazidime cant. Using the data from the antibiotics used singularly, the Hydrate, Sigma life science, C3809–1G, lot: SLBD0920V) independent samples Mann–Whitney U test was employed to and gentamicin (Gentamicin injectable, for IM or IV injection, test that the two antibiotics had the same concentration dis- 80 mg in 2 ml, Gentacin, 051365) was placed in triplicate tribution. Further using this data, both the Pearson’s correla- wells of a gamma-irradiated 96-well U-bottom microtitre tion and Spearman’s rho analyses were used to test whether plate (separate plate for each antibiotic). Dilutions were cre- there was a correlation between the amount of growth visi- ated in the wells using Mueller Hilton broth as a diluent. ble, after the initial incubation, and the strength of the anti- To this, 50 µ l of 1–2 × 10 CFU/ml of the aforementioned bac- biotics. To test whether there was a difference in the MIC terial suspension was added to each well, and each plate had values of antibiotics used singularly, a Chi-squared test was the lid replaced and sealed with tape before being placed in used. To test whether there was a difference in the amount of the incubator at 30°C for 24 h. The MIC was defined as the growth, after the initial incubation, between the five combi - lowest dilution of antibiotic which completely inhibited the nation plates, a chi-squared test was employed. growth of P. fluorescens , with growth determined as a cream/ white dot in the centre of a well (Igbinosa et al., 2012). For Results MBC testing, a loop of solution from wells not containing growth after the initial incubation was spread onto nutrient MIC and MBC tests agar and incubated for a further 22 h at 30°C. The MBC was determined as the lowest dilution of antibiotic in which no For the MIC test for ceftazidime used against Pseudomonas growth was seen on the agar plate, where growth was deter- fluorescens , the MIC was determined as 3 µ g/ml, as it was the mined as cream/white colonies (Igbinosa et al., 2012). lowest concentration to show no growth. For the MBC test 3 Research article Bioscience Horizons • Volume 7 2014 for ceftazidime used against P. fluorescens , the concentra- occurred; thus, clinical recommendations to use ceftazidime tions within the range of 256–3 µ g/ml were plated on nutri- and gentamicin in combination to treat a Pseudomonas infec- ent agar in triplicate. All three triplicate wells containing tion are not due to a synergistic effect. 3 µ g/ml showed growth, and so the MBC was determined as Ratio of antibiotics in combination analysis 4 µ g/ml. For the MIC test for gentamicin used against P. fluo - rescens, the MIC was determined as 0.25 µ g/ml, as it was the There is a statistically significant difference in the amount of lowest concentration to show no growth. Although 1 out of growth between the five combinations of gentamicin and the 3 wells containing 0.75 µ g/ml of gentamicin had growth ceftazidime used, after the initial incubation, with a signifi - present, this was considered as an anomalous result and dis- cance of <0.01 when tested by a chi-squared test. Figure 3 regarded. For the MBC test for gentamicin used against illustrates that when looking at growth after the original P. fluorescens , concentrations 256–0.25 µ g/ml were plated on incubation, as the volume of gentamicin increases and the nutrient agar in triplicate. All three triplicate wells containing ceftazidime decreases, the number of wells showing growth 0.25–2 µ g/ml gentamicin showed growth, and so the MBC decreases. This shows that combinations of 30 µ l gentami- was determined as 3 µ g/ml (Table 1). cin/20 µ l ceftazidime and 40 µ l gentamicin/10 µ l ceftazidime have the greatest bacteriostatic effect against P. fluorescens as As the concentration of both antibiotics increases, when only 21 wells (43%) showed growth in comparison to 35 used singularly, the amount of growth seen decreases; this wells (71%) growth for the combination 10 µ l gentami- illustrates that there is a negative correlation between the cin/40 µ l ceftazidime. The combination 25 µ l gentami- concentration of both antibiotics used singularly and the cin/25 µ l ceftazidime had the least amount of growth return, amount of growth, significant to the <0.01 level, when statis- only one well (2%), when re-plated for the MBC test, illus- tically tested by both the Pearson correlation and Spearman’s trating that this combination had the best bactericidal effect rho. Furthermore, there is a statistically significant difference on P. fluorescens . A combination of 30 µ l gentamicin/20 µ l in the MICs of gentamicin and ceftazidime when used singu- ceftazidime and 40 µ l gentamicin/10 µ l ceftazidime had the larly, when statistically tested with the chi-squared test, with most amount of re-growth when re-plated for the MBC test a significance of <0.01. (20 and 22%, respectively). When looking at the total FICI values for antibiotics in combination amount of growth, growth after the original incubation and further incubation on nutrient agar, the combination of 25 µ l A value of ≤0.5 illustrates synergy; 0.5 < FICI < 4 illustrates no gentamicin/25 µ l ceftazidime has the fewest number of wells, interaction between antibiotics; and ≥4 illustrates antagonism. only 25, showing growth (51%) in comparison to 39 wells Interpreted values will demonstrate whether a synergistic effect (79%) for the 10 µ l gentamicin/40 µ l ceftazidime combina- occurs, giving reason to clinical recommendations to use these tion. The optimal combination of antibiotics comes from the two antibiotics in combination. Plate 1: The FICI values 8.17 25 µ l gentamicin/25 µ l ceftazidime plate, from well F5 where and 4.3 indicate that the two antibiotics are antagonizing each gentamicin has a concentration of 0.5 µ g/ml and ceftazidime other’s antimicrobial activity and hence raising their MIC val- has a concentration of 0.25 µ g/ml, when used against ues. Plate 2: When looking at the FICI values, both 8.04 and 1–2 × 10 CFU/ml of P. fluorescens . 5.3 indicate that the two antibiotics are antagonizing each other’s antimicrobial activity and hence raising their MIC val- Discussion ues. Plate 3: When looking at the calculated FICI value of 2.04, it is illustrated that there is no interaction between the two Pseudomonas fluorescens was susceptible to both gentamicin antibiotics, in that the MIC values remain unaltered by the and ceftazidime as Benito et al. (2012) also found. Extensive other, neither positively nor negatively. Plates 4 and 5: These searches for relevant research regarding these compounds two plates have matching FICI calculations. The well G4 has a and the effect of synergy have yielded little published research calculated FICI value of 4.04 which indicated that the two for P. fluorescens. These results can however be extrapolated antibiotics are antagonizing one another’s antimicrobial activ- to Pseudomonas aeruginosa as P. fluorescens is one of its ity and hence raising the MIC values. However, wells F5 and closest relatives and, in addition, Richards and Brogden E6 have produced FICI values of 2.83 and 1.17, respectively, (1985) and Mikura et al. (2011) state that gentamicin and and this indicates that the antibiotics are neither positively nor ceftazidime are also effective against the more established negatively interacting with one another, to alter the MIC values human pathogen, P. aeruginosa. of one another (Fig. 2 and Table 2). No results of synergy The MIC of ceftazidime was determined to be 3 µ g/ml and Table 1. MIC and MBC results for gentamicin and ceftazidime the MBC to be 4 µ g/ml against P. fluorescen s. This MIC value is within the range of 2–128 µ g/ml as stated by Tunney and Scott (2004) and within the range of 0.5–128 µ g/ml for Antibiotic MIC (µ g/ml) MBC (µ g/ml) P. aeruginosa, stated by Richards and Brogden (1985) and Gentamicin 0.25 3 Rains, Bryson and Peters (1995), who also state that the MBC values are either the same or less than twice the MIC, Ceftazidime 3 4 which the results in this study support. 4 Bioscience Horizons • Volume 7 2014 Research article Figure 2. Pseudomonas fluorescens growth when gentamicin and ceftazidime are used in combination. (A) A volume of 10 µ l of gentamicin and 40 µ l ceftazidime for concentrations ranging from 4 to 0 µ g/ml are in each well-concentrations only shown on (A) but are the same throughout the plates. (B) A volume of 20 µ l of gentamicin and a 30 µ l of ceftazidime of concentrations ranging from 4 to 0 µ g/ml are in each well. (C) A volume of 25 µ l of gentamicin and a 25 µ l of ceftazidime of concentrations ranging from 4 to 0 µ g/ml are in each well. (D) A volume of 30 µ l of gentamicin and a 20 µ l of ceftazidime of concentrations ranging from 4 to 0 µ g/ml are in each well. (E) A volume of 40 µ l of gentamicin and a 10 µ l of ceftazidime of concentrations ranging from 4 to 0 µ g/ml are in each well. To each well, 50 µ l of 1–2 × 10 CFU/ml of P. fluorescens suspension is added. Filled circles represent wells where growth occurred after incubation at 30°C for 24 h. Open circles represent wells where no growth occurred after incubation at 30°C for 24 h. Growth was recognized as a white/cream dot in the centre of the well (MIC test). Wells showing no growth were plated onto nutrient agar for the MBC test. They were incubated at 30°C for 22 h. Circles with cross lines represents re-growth on nutrient agar, recognized as visible cream colonies on the agar (n = 1). The MIC of gentamicin was determined to be 0.25 µ g/ml Gyselynck, Forrey and Cutler (1971) illustrated the ototoxic and the MBC to be 3 µ g/ml against P. fluorescen s. This is effects when high levels of gentamicin are used in vivo. lower than the range of 1–16 µ g/ml for P. aeruginosa stated However, Richards and Brogden (1985) and Rains, Bryson by Burgess and Nathisuwan (2002). The MBC is also lower and Peters (1995) discussed that ceftazidime is eliminated than >8 µ g/ml stated by Rukholm et al. (2006). This could quickly from the body via the urine, and for this reason, it has mean that P. fluorescens is more susceptible to gentamicin limited side effects. Due to the side effects of gentamicin and than P. aeruginosa. However, Sheppard (unpublished data) the quick elimination time of ceftazidime, it would be sensible found the same MIC value as this study, when using the same to believe that using a combination in which the gentamicin stock of gentamicin and same strain of P. fluorescens . level is lowest would be more desirable, for use in vivo, when looking at all of the combinations that produce a desired effect. Safdar, Handelsman and Makim (2004) explain that from conducting a meta-analysis, the use of a combination of anti- Seth and Seth (2009) describe that it is better to treat biotics is more beneficial on mortality rates than using an severe P. aeruginosa infections with a bactericidal antibiotic, antibiotic singularly. During this study, no results of synergy which illustrates that the choice of the 25 µ l gentamicin/25 µ l were produced (FICI ≤ 0.5); however, results of no interac- ceftazidime plate is the best choice, as the lowest amount of tion (0.5 < FICI < 4) were produced. Considering that it is re-growth occurred from this plate, and has a lower volume beneficial to use a combination therapy, the use of the ‘no of gentamicin compared with 30 µ l gentamicin/20 µ l ceftazi- interaction’ results could be used in vivo, with perhaps a ben- dime and 40 µ l gentamicin/10 µ l ceftazidime. eficial effect on the patient, and this study has determined the However, when considering that it is better to treat severe best combination to use. P. aeruginosa infections with a bactericidal antibiotic it would Balakumar, Rohilla and Thangathirupathi (2010) illus- perhaps be beneficial to use combination of antibiotics found in trated the nephrotoxic effects, and Zaske et al. (1981) and well F5 rather than in G5. Well F5 (25 µ l of 0.5 µg/ml gentamicin 5 Research article Bioscience Horizons • Volume 7 2014 and 25 µ l of 0.25 µ g/ml ceftazidime) would produce a FICI of ratio of 1:1, in this case 25 µ l gentamicin/25 µ l ceftazidime, 2.83, which would still demonstrate no interaction between where gentamicin has a concentration of 0.5 µ g/ml and ceftazi- antibiotics. This study shows that it may be best to suggest well dime has a concentration of 0.25 µ g/ml, when used against F5 is more appropriate, because re-growth does not occur at this 1–2 × 10 CFU/ml of P. fluorescens . level, and it is the lowest concentration of gentamicin possible. Antibiotic resistance has become more prevalent in recent Considering this, this study demonstrates that the most practical years, leading to methodologies for treating infection without combination of gentamicin and ceftazidime is to use a volume the concern of antibiotic resistance being needed more than ever. One such method for overcoming antibiotic resistance is Table 2. FICI calculation and interpretation from the checkerboard to use existing antibiotics in combination. The aim of the method results study was to determine whether clinical recommendations to use ceftazidime and gentamicin in combination against Plate Well Calculation Result Interpretation P. aeruginosa are due to a synergistic effect. This study deter- mines that a synergistic effect does not occur, and that this 1 E3 2/0.25 + 0.5/3 8.17 Antagonism recommendation is due to evidence that combination therapy increases a patient’s likelihood of survival compared with a D4 1/0.25 + 1/3 4.3 Antagonism single antibiotic treatment. Although synergy was not found 2 G4 2/0.25 + 0.125/3 8.04 Antagonism in this study, it may prove beneficial to use this combination in vivo. The major limitation of this study is the lack of rep- B5 1/0.25 + 4/3 5.3 Antagonism lication, and this study recommends this treatment be repli- cated in vitro and studied in depth in vivo. 3 G5 0.5/0.25 + 0.125/3 2.04 No interaction 4 G4 1/0.25 + 0.125/3 4.04 Antagonism Author biography F5 0.5/0.25 + 0.25/3 2.83 No interaction A.E.M. has just graduated with a First in BSc Biomedical E6 0.25/0.25 + 0.5/3 1.17 No interaction Sciences from the University of Chester, where during her studies here she gained a passion for research, especially 5 G4 1/0.25 + 0.125/3 4.04 Antagonism within the field of microbiology. For this reason, she is cur - rently studying for a Master of Research (MRes) in Applied F5 0.5/0.25 + 0.25/3 2.83 No interaction Science, at the same university, where her focus is on the E6 0.25/0.25 + 0.5/3 1.17 No interaction Quorum sensing and quorum quenching of Chromobacterium violaceum. Her hopes for the future are to go on to complete Furthest bottom right wells were selected for FICI analysis; in the case of plates 1, 2, 4 and 5, more than one well was chosen for analysis as a staggered a PhD and gain a career in research focusing on antibiotic result was created, as seen in Figure 2. resistance and alternative antimicrobial therapies. Figure 3. The total amount of growth, including original growth, and re-growth, for all five combinations of gentamicin and ceftazidime. G represents gentamicin, C represents ceftazidime. Each well contained a total of 50 µ l of antibiotic and 50 µ l of 1–2 × 10 CFU/ml of Pseudomonas fluorescens suspension. Filled boxes represent the number of wells that showed growth after the original incubation at 30°C for 24 h. Wells that showed no growth were plated onto nutrient agar for the MBC test. They were incubated at 30°C for 22 h. Boxes with lines represent the number of wells where re-growth occurred on nutrient agar (n = 1). 6 Bioscience Horizons • Volume 7 2014 Research article Dempsy, M. (2004) The Approved List of Biological Agents, Advisory Acknowledgements Committee on Dangerous Pathogens, Colegate. I would like to thank my supervisor Dr Neil Pickles for his Donnarumma, G., Buommino, E., Fusco, A. et  al. (2010) Effect of tem - expert advice and support throughout the research process, perature on the shift of Pseudomonas fluorescens from an environ- along with technical staff member Mary Wood for the guid- mental microorganism to a potential human pathogen, International ance she provided for the methodology. Journal of Immunopathology and Pharmacology, 23(1), 227–34. Gershman, M. D., Kennedy, D. J., Nobel-Wang, J. et al. (2008) Multistate Funding outbreak of Pseudomonas fluorescens bloodstream infection after exposure to contaminated heparinized saline flush prepared by a The Department of Biological Science, of the University of compounding pharmacy, Clinical Infectious Diseases, 47(11), Chester, funded this research. 1372–9. References Giamarellou, H. (2002) Prescribing guidelines for severe Pseudomonas infections, Journal of Antimicrobial Chemotherapy, 49(2), 229–33. Aloush, V., Navon-Venezia, S., Seigman-Igra, Y. et  al. (2006) Multidrug- resistant Pseudomonas aeruginosa: risk factors and clinical impact, Guttman, D. S., Morgan, R. L. and Wang, P. W. (2008) The evolution of the Antimicrobial Agents and Chemotherapy, 50(1), 43–8. Pseudomonads, In: M. Fatmi, A. Collmer, N. S. Iacobellis, J. W. Mansfield, J. Murillo, N. W. Schaad, and M. Ullrich (eds) Pseudomonas American Society of Health-System Pharmacists, I. 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Journal

Bioscience Horizons – Oxford University Press

Published: Sep 25, 2014

Keywords: gentamicin ceftazidime Pseudomonas combination therapy synergy

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