Combination Effect of Ciprofloxacin and Streptomycin with Cefotaxime against Multi-Drug Resistant Pseudomonas aeruginosa from Different Clinical Samples

 

Mohammed K. Abbas¹, Duaa A. Kadhum², Adnan Kamel Shabeeb³*, Saif Ali Mohammed⁴

¹Department of Public Health Techniques, Baqubah-Technical Institute, Middle Technical University,

Baghdad, Iraq.

³Department of Medical Laboratories Techniques, Kut -Technical Institute, Middle Technical University, Iraq.

^2,4Department of Medical Laboratories Techniques, Baqubah- Technical Institute, Middle Technical University, Baghdad, Iraq.

 

ABSTRACT:

Purpose: To investigates the synergistic efficacy of synergistic antibiotic combinations effect on P. aeruginosa strains and evaluates the typical antimicrobial effects for Ciprofloxacin and Streptomycin with Cefotaxime on P. aeruginosa strains. Methods: Pseudomonas Isolation Agar (PIA) has been employed to isolate a clinical P. aeruginosa. For minimum inhibitory concentration (MIC) of Cefotaxime, Ciprofloxacin, and Streptomycin, the combination antimicrobial effect of these antibiotics also determined based on half-chess board method. Standard strain of E. coli MM294 E. and E.coli HB101 has been used as control. Results: The clinical samples were resistant to Cefixime Ciprofloxacin by rates of 80%, 5%, respectively. Correspondingly, MICs of the Cefixime, Ciprofloxacin, and Streptomycin were between 32-1024μg/ml, 2–128μg/ml, 128-1224μg/ml respectively. The results of Ciprofloxacin-Streptomycin combination with Cefotaxime showed a synergic effect and a significant reduction in the MIC value at a ratio (1: 3) for 20 strains with percentage of 95.23%, while the other combinations have depicted different fallouts based on synergic, addition, indifference and Antagonism. Conclusions: Cefotaxime with Streptomycin can weaken antibiotic resistance mechanisms of P.aeruginosa strains. It can make the Bacterium, as mentioned above, more susceptible to weaker concentrations of antibiotics.

 

 

 

INTRODUCTION:

Pseudomonas aeruginosa causes hospital-acquired infections owing to its distributive nature, the capability to continue to exist in moist environments, and resistance to numerous antimicrobial agents^[1,2]. The use of two antibiotics from diverse classes may increase the aptness of experiential coverage^[3]. For that reason, the synergism occurs when two or more antimicrobial agents, in combination, employ an inhibitory influence that is bigger than the preservative effects of the alone^[4].

 

The reason to combine more than one antibiotic is to increase the action of these antibiotics, decrease the side effects as well as decrease the dose when conditions of resistance development and indifferences of single antibiotic are dominant in the life-threatening treatment infections^[5]. The antibiotics combination resistance is as a result of numerous factors like constitutive resistance to antibiotics due to the cell wall permeability, mutations in bacterial chromosomes that regulates the expression of resistance genes, and gaining of resistance genes like plasmids, transposons, and bacteriophages^[6]. The synergy between dual antimicrobial agents in vitro provides a efficacious treatment to treat the resistant strain and regulate developing of resilient life-threating infections^[7,8]. The checkerboard or fractional inhibitory concentration (FIC) index is one of multi method that has two or more antimicrobial combination to exhibiting synergism effect. The methodology of (FIC) is analogous to determined (MICs) and the synergism appears in the case of a 4-fold decrease in the MIC value for each antibiotic tested alone^[9].

 

Accordingly, our study aims to examine antimicrobial activity and estimate the interaction of Ciprofloxacin and Streptomycin with Cefotaxime combination against P. aeruginosa. It is thought that the results may provide a rational basis for the clinical use of these combinations against multidrug-resistant P. aeruginosa.

 

METHODS:

Samples Collection:

A total of 180 samples had collected from patients with (20–65 years old) between March to April 2019, attending to Baqubah Teaching Hospital and Bald Ruz General Hospital on Diyala province – IRAQ. These samples have been collected from urologic catheters, vaginal swabs, surgical intramuscular tools, urinary tract infections, and wound infections.

 

Isolation and Bacterial Identification:

All samples and swaps cultured on Pseudomonas selective agar (CHROMagar, French) and 5% Blood agar (Himedia, India) and after that, incubated under 5% CO2 atmosphere at 37C˚ for 18-24 hrs. Conventional Biochemical tests have employed according to McFadden^[10], in addition to the VITEK-2 system (Biomerix, French). Two standard strains MM294 E. coli. and HB101 E.coli were obtained from the Institute of Genetic Engineering, University of Baghdad-Iraq.

 

Antibiotic susceptibility Test:

Antibiotics sensitivity was done based on Kirby – Bauer method by selecting 14 common antibiotics included Cefixime, Ceftazidime, Piperacillin, Cefotaxime, Imipenem Aztreonam, Gentamycin, Amikacin, Tobramycin, Ampicillin, Ciprofloxacin, Nitrofurantoin and Co-Trimoxazol, and Augmentin (Bioanalyse, Turkey), While the Minimum Inhibitory Concentration (MICs) was done using Macro-dilution technique, according to NCCLS^[11].

 

Antibiotics combination:

All P. aeruginosa isolates have tested with their Synergistic ability based on half-chess board method that clarified by^[12], through mixing Ciprofloxacin and Streptomycin with Cefotaxime. The concentration is (1: 0.5, 1: 1, 1: 2, 1: 3). The fractional inhibitory concentration (FIC) was derived from the lowest concentration of antibiotic combination allowing invisible growth of the tested organism in the tube calculated for each antimicrobial concentration as follows: FIC of compound A (FIC A) = MIC of compound A in combination with B / MIC of compound A alone. FIC of compound B (FIC B) = MIC of compound B in combination with A / MIC of compound B alone. The sum of FIC index = FIC A+FIC B. Combination between antibiotics according to^[13] is as following: ≤ 1.5, synergy; 1.5 to 1.1, additive; 1.1 to 4.1, indifference; and > 4, antagonism.

 

RESULTS:

About 180 different samples collected from 50 surgical operations swabs, 30 Urine samples, 50 vaginal swabs, and 50 Intrauterine swabs. About 100 (55.5%) samples have given negative bacterial growth, and 80 have shown bacterial positive growth, but 21 isolates (26.2%) are belonging to P.aeruginosa. [Table 1] explains the details of isolates.

 

Table 1: The obtained percentage of samples and isolates

 

P.aeruginosa isolates were identified by their Morphologic, Microscopic, and Biochemical characteristics showed in [Table 2]. The API 20NE (Biomerix, French) has been used to be confirmative.

 

Table 2: Biochemical tests used in this study

Negative results (-), Positive results (+), Variable (v).

 

For the antibiotics sensitivity test, our study have depicted high resistance in contradiction of Cefixime, Ceftazidime, Cefotaxime, Aztreonam, Piperacillin, and Ampicillin with percentages of (80%, 75%, 60%, 60%, 70%, and 70%) respectively. The antibiotics sensitivity has been illustrated in [Figure 1].

 

Figure1: The Percentages of antibiotic sensitivity test

 

The results of Multi-Drug resistance have shown that 20 isolates (95.23%) had multi-resistance against 3 to 13 Antibiotics out of 14 Antibiotics used in the study as detailed in [Table 3].

 

Table 3: Multi- Drug resistance distribution between isolates

P =P.aeruginosa

 

The tube dilution assay (MIC test) has been explained in [Table 4]. The Concentrations of Cefotaxime was between 32-512μg/ml for P.aeruginosa isolates, while for Streptomycin, the result was between 128-1024μg/ ml concerning Ciprofloxacin. Our result was in the range between 2-1024μg/ml.

 

In this study, Cefotaxime has a persistent activate ingredient in all mixing ratios, and a better mixing ratio has been determined. Cefotaxime- Ciprofloxacin combination (1: 3) was the best mixture for 20 isolate. (Figures 2,3,4,5,6) showed a significant reduction in the MIC ranges of antimicrobials after the combination of Ciprofloxacin and Streptomycin with Cefotaxime than in the case of each antibiotic alone.

 

Table 4: Minimum Inhibitory Concentrations (MIC).

 

The effect of Ciprofloxacin - Cefotaxime combination with ratio (1: 0.5) was with 4 isolates (19%) showing synergistic effect while 6 isolates have been indifferent in this mixture. Also, 4 isolates have shown supplementary effect to the mixture, 7 (43%) isolates have shown Antagonism effect. For (1: 1) ratio, 9 isolates (42.8%) have shown synergic effect, 6 isolates indifference, 3 isolates addition effect and 3 isolates Antagonism effect (Figure2,3).

 

For a ratio (1: 2), 11 isolates (52.38%) showed a synergetic effect, 2 Antagonism effect, 8 isolates showed addition effect (Figure 4 a). for a ratio of (1: 3), 20 isolates (81%) have shown Synergetic effect, 1 isolate have shown additional effect, while 2 have indifferent effect as depicted by (Figure 4 b).

 

Figure 2: The MIC of Ciprofloxacin - Cefotaxime Combination with ratio (1: 0.5)

 

Figure 3: The MIC of Ciprofloxacin - Cefotaxime Combination with ratio (1: 1)

 

(a)

 

 

(b)

Figure 4: The MIC of Ciprofloxacin - Cefotaxime Combination with (a) ratio (1: 2) and (b) ratio (1:3)

The Streptomycin (Aminoglycoside group) was the second antibiotic to combine with Cefotaxime. The effect of their mixing has been in a ratio (1:0.5). (Figure 5 a) depicts a synergistic effect for 11 isolates (52.3%), 4 indifferences, 4 additions, and 2 with Antagonism effects.

 

(a)

 

(b)

Figure 5: The MIC Value of Streptomycin- Cefotaxime Combination with (a) ratio (1: 0.5) and (b) with ratio (1: 1).

For a ratio (1: 1), a synergistic effect was appeared in (10) isolates (47.6%), 9 isolates with addition effect, 1 isolate showed Antagonism effect and 1 isolate showed Indifference in activity based on (Figure 5 b).

For a ratio (1: 2), the results showed a synergic impact for 13 isolates (61.8%), 6 Addition, 1 isolate showed indifference effect (Figure 6 a).

 

 

(a)

 

(b)

Figure 6: The MIC Value of Streptomycin- Cefotaxime Combination with (a) ratio (1: 2) and (b) ratio (1: 3).

 

The best mixing ratio was (1:3) for the reason that 20 isolates showed synergic effect excluding 1 isolate showed an addition effect based on (Figure 6 b).

 

DISCUSSION:

P.aeruginosa stands for a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A significant factor in its distinction as a pathogen is its intrinsic resistance to common antibiotics and disinfectants that the escalating rates of resistance to antibiotics left clinicians with limited choices to treat severe infections^[14]. Multidrug resistance bacteria (MDR) is the resistance versus three classes of antimicrobial and the resistance has increased in recently^[27,28]. Most of the antibiotics were effective in contradiction of P. aeruginosa except ampicillin, Aztreonam, Imipenem, and Cefixime. Antibiotics resistance can either be inherent or acquired by the genetic mutation or resistance gene transfer^[15]. The outcome of this combination is agreed with^[16,17] who demonstrated that Cefotaxime can be an effective choice against P.aeruginosa in vitro but this differ completely with^[18]. Who found the percentage of P.aeruginosa resistance to Cefotaxime with (90.4%). With regard to Ciprofloxacin, our result was converge with reported papers in^[19,20]. The resistance of Quinolones, especially Ciprofloxacin, is due to in effect ciprofloxacin on the determination of the gyrase DNA responsible for the superlative association of DNA and IV topoisomerase and the occurrence of mutations in Chromosomal DNA in P. aeruginosa. Hence, 8 isolates were resistant more than dual antibiotics. This acquired resistance, apart from being related with numerous mutations in topoisomerase encoding genes, is predominantly associated with efflux systems. Their down regulation or up regulation (depending on whether the regulator is positive or negative) contributes significantly to the reduced activity of fluoroquinolones^[21]. Combination therapy with both antimicrobials are suggested to treat treating life-threatening infections for indicated relationship between the MIC and clinical outcome of infections due to P.aeruginosa treated with a single antimicrobial. Specifically, a lower MIC has associated with a faster healing response^[18]. The MIC for Cefotaxime was 32-512μg/ml. This result converges with study^[22], who demonstrated that MIC was within 8-512 μg/ml range and differed with study^[23], who found that MIC was between (1-64 μg/ml).

 

With regard to Ciprofloxacin, our result was converged with^[19]. in which the MIC was demonstrated between (8-512) In the checkerboard method, the interaction between selected combinations of the four antibiotics against 21 isolates of P. aeruginosa as estimated by the mean FIC index values are depicted in figures (3,4,5,6) for P. aeruginosa. We perceived that the combination of ciprofloxacin- Cefotaxime acts synergistically for increasing concentration of the antibiotics and shows strong bactericidal activity against P. aeruginosa. In vitro synergetic effect of the cefotaxime- ciprofloxacin combination against enteric, Gram-negative Bacteria has been reported for E. coli^[24], Serratia^[25], and Salmonella Typhi and Paratyphi.^[26]. this study examines the effect of antibiotic combinations therapy against P.aeruginosa infection with lot of limitations. Firstly, lack of isolation and increased numbers of isolates contribute to the analysis of ranges of resistance and sensitivity to the standard antibiotics. The results have depicted that the synergistic effect is limited but much better than the use of these antibiotics alone. It may also don't fit with high P.aeruginosa virulent. Consequently, Synergistic results of ciprofloxacin or Streptomycin - Cefotaxime make this combination beneficial. Nonetheless, further analysis of P.aeruginosa in a clinical setting is needed to evaluate the interactions of this combination.

 

CONCLUSION:

The combinations of Ciprofloxacin and Streptomycin –Cefotaxime have given the lowest mean FIC index for P.aeruginosa and synergistic effect in the ratio (1:2, 1:3), while other tested combinations were predominately additive or indifferent. In this study, the combination is efficient against the isolates under investigation, and the synergistic effect predominates obviously.

 

ACKNOWLEDGMENTS:

Acknowledgments of technical help from assistant lecturer Saif A. Mohammed master in the science of Microbiology and general support from the departmental chair to provide a place work antibiotic tests.

 

CONFLICTS OF INTEREST:

There are no conflicts of interest.

 

CONTRIBUTION OF AUTHORS:

We declare that this work was done by the authors named in this article and all liabilities Pertaining to claims relating to the content of this article will be borne by the authors.

 

The manuscript was not presented as part at a meeting, the organization or another place.

 

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Received on 16.01.2020          Modified on 14.03.2020

Accepted on 27.04.2020         © RJPT All right reserved