In vitro antimicrobial activity of generic and brand-name Levofloxacin against clinical and ATCC strains E . coli and S . aureus

In vitro antimicrobial activity between generic and brand-name Levofloxacin was evaluated against isolated strains collected in 3 Colombia hospitals: Staphylococcus aureus and Escherichia coli. Initially, active substance was quantified using the methodologies identified by the United States Pharmacopeia (USP) 38 NF 32, chromatographic conditions were validated and standardized. The minimum inhibitory concentration (MIC) of Levofloxacin was determined in accordance with the Clinical and Laboratory Standards Institute (CLSI). Growth curves were then performed to determine the maximum growth time of the bacteria in order to determine the MIC at the maximum growth time. Different brands evaluated did not present any difference with MIC of 0.125, 0.062, 0.031, 0.062 and 0.125 μg/ml for E. Coli ATCC, E. Coli Tropical, S aureus sensible ATCC, S aureus resistant ATCC and S. luteum, respectively. The in vitro antibacterial activity of levofloxacin against E coli Tropically and S luteum are reported for the first time.


INTRODUCTION
Fluoroquinolones are the fourth class of antibiotics used in human and veterinary medicine for the treatment of serious bacterial infections.Its broad spectrum of activity and favorable pharmacokinetic properties are the main characteristics that have increased its widespread clinical use throughout the world (Barreto et al., 2017).However, its irrational use has increased the resistance profile, for this reason, it is pertinent to conduct studies that evaluate pharmaceutical alternatives of Levofloxacin, against pathogenic microorganisms such as S. aureus and E. coli sensitive and resistant, as well as ATCC isolated from hospitals in Colombia (Carvalho et al., 2016;Fariña et al., 2007).Currently, the doubts that arise both in the users and health providers services are very evident when it comes to the quality of any generic drug, and their replacement with brand name drugs (Artaza et al., 2016;Medina-Morales et al., 2015).For these reasons, it is necessary to demonstrate that the quality of a drug is not directly related to its value and to dispel the myth "the more expensive the product, the more effective", which may favor communities with less economic capabilities, allowing them access to effective and quality therapy, which until now would be the most affected due to the few numbers of studies.
Bacterial resistance to antibiotics is a global health problem, because the possibility of continuing to successfully treat infections that are now easily treated is in danger (Yılmaz and Aslantaş, 2017).Morbidity, mortality and treatment costs will increase, if these bacterial resistances are not controlled (Pastor-Sánchez, 2006;Jackson et al., 1998;Juste Díez de Pinos et al., 2000;Mandell et al., 2010).The irrational use of antibiotic therapy jeopardizes the possibility of continuing treating with success, infections that are treated with these drugs; to clear up the doubts, we must evaluate the behavior of these substances and certify the suitability of the products used for the therapies indicated, guaranteeing the interchangeability between generic Levofloxacin with its brand name in the treatment of the pathologies caused by E. coli and S. aureus.
For all drug generic or brand name, especially antibiotics, their efficacy and safety are infallible qualities, since in the opposite case; the patient health is put at risk due to the appearance of bacterial resistances (Sun et al., 2016).According to the surveillance programs in United States, a total of 2008 samples evaluated showed a resistance rate to Levofloxacin of 5% (Cercenado, 2011;Meléndez et al., 2005;Sato et al., 2011).
The aim of the study is to compare in vitro antibacterial activity of a generic and brand name of Levofloxacin previous substance active quantification, following USP38 NF 32 (Pharmacopeia, 2016).The activity of Levofloxacin was measured against 2 strains which are causes of nosocomial infections: Escherichia coli and Staphylococcus aureus, by determining the minimum inhibitory concentration (MIC), using broth microdilution method.The results obtained allow us to determine which drug offers the highest efficacy in vitro.In addition, this study will present results in strains which have not been evaluated microbiologically in Colombia: Escherichia coli tropically and Staphylococcus luteum.

Chromatographic conditions
The high resolution liquid chromatography (Elite Lachrom HITACHI I2350) equipment, equipped with a quaternary pump and a Diode Array Detector (DAD) was used.A reversed phase Merck® C18, 150 × 4.6 mm, particle size 5 μm was used as the analytical column.The mobile phase was a mixture of 0.1% solution of triethanolamine-acetonitrile (80:20), adjusted to a pH of 4.8 with phosphoric acid; filtered and degassed by 0.45 μm membrane.The wavelength was set at 296 nm, with flow rate of 1 ml/min and injection volume of 20 μl.Before using all solutions, the mobile phase was sonicated for 30 min and UV detection was performed at 296 nm for GTX.
Linearity 10 mg of Levofloxacin standard were weighed and taken to a 10 ml graduated volumetric flask, which was completed using mobile phase diluent, thus remaining at a concentration of 1000 ppm.This solution was labeled as standard stock solution.From the stock solution, aliquots of 0.25, 0.5, 0.75, 1.0, and 1.2 ml were taken to 10 ml graduated volumetric flask and adjusted with mobile phase, obtaining concentrations of 25, 50, 75, 100 and 120 ppm, respectively.Each solution was injected into the Chromatograph in triplicate.

Accuracy
Two milliliter of the drug Levofloxacin was taken, which was at a concentration of 5 mg/ml and taken to a 10 ml graduated volumetric flask, it was completed using mobile phase diluent, obtaining a concentration of 1000 ppm.It was labeled as the stock solution.
From the stock solution, aliquots of 0.25, 0.5, 0.75 and 1.0 ml were taken to 10 ml graduated volumetric flask and adjusted with mobile phase, obtaining concentrations of 25, 50, 75 and 100 ppm, respectively.Each solution was injected into the chromatograph in triplicate.The obtained data were analyzed and the recovery percentage calculated.

Repetitiveness
From the stock standard and sample solutions, 1 ml aliquots were taken to 10 ml graduated volumetric flask; mobile phase was adjusted and 100 ppm concentrations were obtained.These solutions were taken to the chromatograph and injected six times each.The obtained data were analyzed and the standard deviation and the coefficient of variation (RSD) were calculated; having an RSD ≤ 2% as acceptance criteria for the runs.

Intermediate precision
From the standard stock solution aliquots of 0.5, 0.75 and 1 ml were taken to 10 ml graduated volumetric flask, to which volume was completed with mobile phase and a solution was obtained with concentrations 50, 75 and 100 ppm, respectively.These solutions were injected in duplicate.This procedure was carried out by three different analysts on different days.The data obtained were analyzed and the relative standard deviation (RSD) obtained.

Antimicrobial activity
The minimum inhibitory concentration (MIC) of 2 generic, 1 USP standard and 1 brand name of levofloxacin drug were evaluated using the broth micro dilution method, described by the Clinical and Laboratory Standards Institute (CLSI, 2015).Ten dilutions were prepared for each drug, performing serial double dilutions from 64 to 0.0075 μg/ml on bacterial suspensions at a concentration of 5 × 10 5 CFU/ml, in 96-well microtiter plates CLSI (2011).Initially beginning with a concentration of levofloxacin drugs (5 mg/100 ml) which was diluted with Muller-Hinton broth at pH 7.3 to obtain a stock solution of 64 μg/ml, the solution was diluted to obtain an intermediate solution at a concentration of 8 μg/ml after which, doubling-dilution series of the antibiotic solutions of 8 μg/ml to 0.015 μg/ml were performed.50 μl of each dilution was dispensed into the wells of the Microplates and 50 μl of the inoculum was added to each one, to obtain final bacterial concentrations of 5 × 10 5 CFU/ml.A well that contains inoculum without antibiotic was used as a positive control and one containing antibiotic dissolved in broth without inoculum as a negative control.The turbidity of the actively growing broth culture was adjusted to an optical density equivalent to a 0.5 McFarland standard using a Thermo Scientific Multiskan EX® spectrophotometer at 620 nm.All assays were conducted in triplicate.

Statistical analysis
The analytical datas, such as linearity, accuracy, repetitiveness and intermediate precision, were tested for each alternative through descriptive statistics.MIC values between doubling-dilution series of the antibiotic solutions, positive control and negative control for each alternatives, were tested using one-way analysis of variance (ANOVA), followed by a tukey test for multiple comparisons with significant statistical difference at p < 0.05.

Linearity
The results obtained indicate that the system for determining levofloxacin is able to explain the response (Area) from the use of the concentration variable.Therefore, in the concentration range between 25 and 120 ppm the linearity conditions of the analytical system are satisfied, this is demonstrated by obtaining a correlation coefficient r = 0.9982 and a determination coefficient R 2 = 0.9965.

Accuracy
Table 1 shows the levofloxacin recovery percentage values, which reached between 94.64 and 100.83%, which are within the acceptance criteria of 92% as a minimum.The values of RSD remained below 2%, this

Repetitiveness
The recovery percentage for each injection were calculated, and average values of 93.31% for the system and 93.79% for the method were obtained, indicating that the method and system met the requirements to perform the test (Tables 2 and 3).

Intermediate precision
The RSD values obtained were between 0.01% and 0.05%.Likewise, the calculation of the recovery percentage was made for each run; the average value obtained was 102.58% (Tables 4, 5 and 6), this is due to analyst linked errors during the preparation of the solutions, evidenced when finding the real concentration of these solutions.These results show that the analytical method is accurate, since the USP38 accepts as a minimum value or acceptance criterion for this parameter, an RSD less than or equal to 4% and a recovery percentage greater than or equal to 95%.

Generic and brand name comparison
The comparison test between generic and brands name levofloxacin drug yielded very similar results in terms of areas under the curve and the recovery percentage, with an average of 97.76% for the generic drug and 97.11% for the commercial one.Both cases had a variation coefficient (RSD) of 0.01.The generic and brand name drug vials concentration were determined using the formula described in the methodology, obtaining a concentration of 4.861 mg/ml for the generic drug and 4.826 mg/ml for the brand name one (Tables 7 and 8), corresponding to 97.22% for the generic drug and 96.52% for the brand name one of the reported concentration (5 mg/ml).In this study, the analytical quantification of active principle of generic and brand names levofloxacin shows that there were no differences from the point of view of the concentration reported in the tag of the different drugs evaluated.This is directly    S. aureus, 18 h E. coli, showing a time of maximum growth greater than ATCC strains, previously evaluated and presenting no abnormalities with respect to growth.It is possible to observe and differentiate the stages from medium assimilation that is less than 5 h to the stage of death, which in the case of S. aureus is between 18 and 20 h, and for E. coli it is observed from 19 h.Hence, the timing of maximum growth of the isolates is greater than ATCC.Comparing Microkit SL laboratories strains to those isolated from hospitals and to ATCC, it was observed through absorbance performed by turbidimetry tests, that hospital strains had the highest cellular concentration over time of maximum growth (Figure 2).Although Microkit SL laboratories strains obtained the longest time of maximum growth, they presented less bacterial concentration.Thus, comparing hospital isolated strains; it can be concluded that those obtained from ICU had a higher bacterial concentration, in a smaller time unit.The MIC values of standard, generic and brand name of levofloxacin are presented in Tables 9 and 10.
Overall, the MIC of different levofloxacin drugs against S. aureus and E coli evaluated were found much lower than that reported by Martínez et al. (2004) and Van Bambeke (2005).On the other hand, MIC of levofloxacin was determined against to E. coli tropically and S. luteums, for the first time in Colombia.None of the strains exceeded the ranges reported by literature.However, we must take into account the significant difference between MIC values presented by Clinical Isolated strain of Colombian hospital, which were higher with respect to ATCC and MICROKIT SL laboratory strains.Although, they do not exceed the limits established by the above referenced studies, this outcome could be in relation with the increase of levofloxacin resistance (Kao et al., 2016), reason for which its use in clinic has decreased in Colombian Hospital.
Table 11 shows the results of confirmative tests confirming that the data obtained by turbidimetric method used in the present study were correct.Furthermore the MIC values remained the same as in the previous trials, confirming the low efficacy against clinical isolates compared with standard strains of E coli and S aureus for both generic and brand name of levofloxacin

Conclusions
No significant differences existed in active principle substance concentration between the different brands of levofloxacin evaluated by a precise, repetitive and accurate method.This is directly related to the quality of levofloxacin used for in vitro antibacterial activity assay.When comparing the differences between generic and brand name levofloxacin, the differences in MIC values were very minimal.It was possible to demonstrate that generic and brand names levofloxacin showed similar in vitro antimicrobial activity against Clinical and ATC strain of S. aureus and E. coli.However the clinical isolation strain presented MIC to be more elevated that ATCC strain for both generic and brand name levofloxacin, this outcome could be the relationship with the increase of levofloxacin resistance, and this would explain the low efficacy of quinolones in clinic.This study reported for the first time the levofloxacin MIC against two specific strains from Colombia that had never been evaluated against E. coli tropically and S. luteum.

Figure 1 .
Figure 1.Recovery percentage between generic and Brand name levofloxacin drugs.

Figure 2 .
Figure 2. Growth curves A. S aureus sensitive ATCC B. S aureus resistant, ATCC C. E coli ATCC.D. S luteum, E. E coli Tropically, F and G. S aureus and E coli clinical isolated.

Table 1 .
Percentage of recovery of Levofloxacin estimated by precision test, USP 38.

Table 2 .
Percentage of recovery of Levofloxacin estimated by system repeatability test, USP 38.

Table 3 .
Percentage of recovery of Levofloxacin estimated by method's repeatability test, USP 38.

Table 4 .
Percentage of recovery of Levofloxacin estimated by intermediate precision test, day 1, USP 38.

Table 5 .
Percentage of recovery of Levofloxacin estimated by intermediate precision test, day 2, USP 38.

Table 6 .
Percentage of recovery of Levofloxacin estimated by intermediate precision test, day 3, USP 38.

intermediate precision day 3 Retention time Área Theoretical concentration (ppm) Real concentration (ppm) Recovery %
E. coli and S. aureus ATCC strains, both sensitive and ; Cattaneo et al 2009).On the other hand, E. coli tropically and S. luteum canariensis strains reached up to 23 h, which is also compatible with that reported by Microkit SL laboratories in the technical annexes provided.Microkit laboratories strains grow faster in time than ATCC strains.Hospital strains presented the following timing of maximum growth; 16 h

Table 7 .
Percentage of recovery of Levofloxacin generic drug, USP 38.

Table 8 .
Percentage of recovery of Levofloxacin brand name USP 38.

Table 9 .
Standard, generic and commercialLevofloxacin MIC in the maximum growth time for each strain.MICROKIT SL laboratory strains, **Clinical Isolated strain. *

Table 10 .
Standard, generic and commercial levofloxacin MIC, 24 hours after the maximum growth time for each strain.

Table 11 .
Results of all confirmative tests.