Pneumonia is the single largest infectious cause of death in children worldwide and also a form of an acute respiratory infection that affects the lung. The purpose of the study was to develop a new approach to treat antibiotic-resistant K. pneumoniae infection. This study aimed in quest of a drug to combine with ciprofloxacin, a broad spectrum antibiotic frequently used to treat lung infections. Methodology: A total of 23 lung infection bacterial samples were collected and studied against 14 antibiotics of different classes. The disk diffusion method was performed to determine synergy screening, MIC value, and qualitative toxicity analysis of ciprofloxacin and chloramphenicol combination. Results: After primary screening of antibiotic susceptibility, they were categorized into multidrug-resistant (MDR), extensively drug-resistant (XDR) and pan drug-resistant (PDR) pathogens where 9 isolates were MDR, 5 were XDR and 3 isolates were PDR. Furthermore, they were trialed in combination ciprofloxacin along with other 7 drugs in disk diffusion to explore the synergistic effect. The combination of ciprofloxacin and moxifloxacin, ciprofloxacin and chloramphenicol were found to be synergic. Then the MIC test was done for the combination ciprofloxacin and chloramphenicol. When the MIC result was generated, the MIC of the respective combination was analyzed. Furthermore, the fractional inhibitory concentration (FIC) was calculated and in accordance with the results of the FIC index, ciprofloxacin-chloramphenicol combination has shown value 0.4510 which revealed a synergistic effect against multi-drug resistant Klebsiella pneumoniae. Conclusion: Given these points, if the efficiency of this antibiotic can be accelerated from combination with other drugs, it might be lifesaving and cost effective as well.
Pneumonia has become the leading cause of child death since many decades in developing countries [
The emergence of antibiotic resistant microorganism is natural and genetically over time. Still there are certain acceleration factors yielding the development of resistant strain. According to World Health Organization (WHO) the overuse and misuse of frequent antibiotic is the culprit behind the resistant mechanism [
Klebsiella pneumoniae carbapenemases (KPCs) were identified in the USA in 1996 [
Ciprofloxacin consists of 8 different functional groups (shown in
However, physicians start using timely antibiotic combination therapy to improve the patient survival since the antibiotic treatment for these resistant bacteria is limited [
The experiment was carried in the laboratory of BRAC University to study the activity of ciprofloxacin having combination with several drugs against multidrug resistant Klebsiella pneumoniae. The experiment proceed mainly involved in collecting the pathogenic Klebsiella pneumoniae and their antibiotic susceptibility testing. Then, the samples were categorized into Multidrug resistant (MDR), Extensively Drug resistant (XDR) and Pan Drug resistant (PDR) on the basis of their susceptibility to 14 different antibiotics including Penicillin G, Penicillin V, Ampicillin, Amoxicillin, Cefixime, Imipenem, Cephalosporin (Cell wall synthesis inhibitor); Chloramphenicol, Tetracycline, Azithromycin (Protein synthesis inhibitor) and Ciprofloxacin, Nalidixic Acid, Rifampicin and Moxifloxacin (Nucleic acid synthesis inhibitor). Randomly 2 MDR, 2 XDR and 2 PDR pathogen are picked for the activity study of several drugs combined with ciprofloxacin. In this study, 8 different drugs of various groups are combined with ciprofloxacin disc to demonstrate their combined effect. The combination of chloramphenicol with ciprofloxacin is assumed to be efficient from exploring the zone of inhibition data result. To be clear with the assumption, the Minimum Inhibitory Concentration (MIC) of these combinations was figured out along with the MIC of individual one. Finally, the Fractional Inhibitory Concentration (FIC) index of each combination (Except the combination of ciprofloxacin and probiotic) was calculated and compared with the standard for statistical validation.
Clinically identified Klebsiella pneumoniae was collected from the National Institute of Diseases of the Chest and Hospital (NIDCH) and microbiology department of Uttara Adhunik Medical College Hospital (UAMCH).The isolates of
Klebsiella pneumoniae was sub-cultured to nutrient agar slant and carried to BRAC University laboratory. The nutrient agar slant was incubated at 37˚C for 24 hours. Then the pathogen was transferred to nutrient agar plate by streaking plate method.
At the same time these samples are stored at −20˚C in glycerol media as stock.
Ciprofloxacin was taken from the product of Square Pharmaceuticals, Ciprocin 500 mg. The other drugs for combination purpose are listed in
Commercially available tablet or capsule was bought and dissolved in 10 ml physiological saline. Though the excipients of those drugs were also inside the solution, it was considered not to be interfered with the desired product since excipients are chemically inert.
Tablet or capsule was poured in physiological saline. As a result, the volume of the 10 ml saline raised very little which was negligible and excluded from the calculation.
Agar surface of Muller-Hinton Agar plate was streaked by a sterile cotton swab with the collected pathogenic Klebsiella pneumoniae strain from the physiological saline which was compared with McFarland standard 1 solution. McFarland standard 1 solution shows the density of 3 × 108 CFU (Colony Forming Unit) per ml. Antibiotics discs were placed on solidified agar plates at equal distance apart. The plates were kept standby for 10 min. Then the plates were incubated at 37˚C for 24 hours. The disc diffusion test was done to determine the antibiotic resistant pattern of the pathogens as well as to categorize to MDR, XDR and PDR by the guideline of Clinical and Laboratory Standards Institute (CLSI). Around 14 antibiotic discs were used in this study.
| Trade Name | Company Name | Generic Name | Class |
|---|---|---|---|
| Fexo 120 mg | Square Pharma | Fexofenadine | Antihistamine |
| Rifagut 200 mg | Opsonin Pharma | Rifaximin | Miscellaneous Antibiotic |
| Indever 10 mg | ACI Limited | Propranol Hydrochloride | Calcium channel blocker |
| Cloram 5 mg/ml | Ibn Sina Pharma. | Chloramphenicol | Antibiotic |
| Tycil 500 mg | Beximco Pharma | Amoxicillin | Antibiotic |
| Moxibac | Popular Pharma | Moxifloxacin | Antibiotic |
| Ciprocin | Square Pharma | Ciprofloxacin | Antibiotic |
The study was designed to observe the efficiency of ciprofloxacin alone and with combination against highly antibiotic resistant Klebsiella pneumoniae. Hence, the MIC of ciprofloxacin and the MIC of other individual antibiotics were figured out with or without combination.
Determining the MIC, different concentration of antibiotic was required. For this purpose, serial dilution was carried out with the aid of Brain Heart Infusion (BHI) broth as diluent. From the C1V1 = C2V2 formula, desired concentration was prepared by the addition of stock solution to BHI broth.
A wide range dilution was prepared with individual antibiotics and combination as well. Throughout the study, it was practiced to keep the concentration of different compounds same. Each test tube having a known concentration of antibiotic was inoculated with 100 µL of McFarland 1 standard pathogenic suspension and kept at 370 Celsius for 24 hours. The next day, each tube was critically observed to identify either turbid or clear. The lowest concentration of antibiotic gave clear tube was considered as MIC value.
The MIC test was done twice with different dilution range to get more sophisticated result through arithmetic mean calculation.
Fractional Inhibitory Concentration (FIC) index is a statistical tool for validation. The standard value of FIC index is 0.5 to 4. The lower value represents the synergism and higher value for antagonism.
To get FIC index, firstly, FIC was calculated by the following equation:
FIC = MIC of the agents in combination MIC of the agent alone
FIC index was calculated by the formula of:
FIC Index = ∑ MIC of the agents in combination MIC of the agent alone
Synergy is defined as ƩFIC ≤ 0.5, indifference is defined as 0.5 < ƩFIC ≤ 4, and antagonism is defined as ƩFIC > 4 [
The average FIC index from six Klebsiella pneumoniae was determined and compared to standard.
When a species of microorganism shows non-susceptibility to at least one agent in three or more antimicrobial categories it is classified as MDR. When this non-susceptibility is shown against at least one agent in all but two or fewer antimicrobial categories it is classified as XDR. PDR is defined when non-susceptibility is shown to all agents in all antimicrobial categories
MDR = Resistant to Penicillin G, Penicillin V, Ampicillin, Amoxicillin, Ciprofloxacin, Nalidixic acid, Chloramphenicol.
XDR = Resistant to Penicillin G, Penicillin V, Ampicillin, Amoxicillin, Cephalosporin, Cefixime, Chloramphenicol, Tetracycline, Nalidixic acid, Ciprofloxacin.
PDR = Resistant to all 14 antibiotics.
A. The antibiotic susceptibility test result of Ciprofloxacin + Amoxicillin combination against Klebsiella pneumoniae (left picture). B. The antibiotic susceptibility test result of Ciprofloxacin + Rifaximin and Ciprofloxacin + Fexofenadine combination against Klebsiella pneumoniae (right picture) shown in
Two MDR, two XDR and two PDR were selected randomly from the collected Klebsiella pneumoniae pathogen in an effort to inhibit the growth of the pathogen by ciprofloxacin having various combinations with 7 different drugs including antihistamine, antibiotic, calcium channel blocker and probiotic which are shown in
Since these pathogens were multidrug resistant, the MIC value of ciprofloxacin and chloramphenicol was pretty high and the MIC value of the combination was nearly low as shown in
The arithmetic mean of FIC index is 0.4510 which is less than 0.5 yielding statistical significant synergistic effect of ciprofloxacin and chloramphenicol against the multiple antibiotics resistant Klebsiella pneumoniae.
| Total Sample | MDR | XDR | PDR |
|---|---|---|---|
| 23 | 9 | 5 | 3 |
| Combination of Drugs | Inhibition of Growth |
|---|---|
| Ciprofloxacin + Chloramphenicol | + |
| Ciprofloxacin + Amoxicillin | - |
| Ciprofloxacin + Rifaximin | - |
| Ciprofloxacin + Propranol Hydrochloride | - |
| Ciprofloxacin + Fexofenadine | - |
| Ciprofloxacin + Moxifloxacin | + |
| Ciprofloxacin + Clonazipum | - |
Key: (+) = synergic, (-) = No change.
| Category | Sample Number | MIC (in µm/ml) | FIC Index | ||
|---|---|---|---|---|---|
| Ciprofloxacin Only | Chloramphenicol Only | Ciprofloxacin + Chloramphenicol | |||
| MDR | Sample 2 | 425 | 225 | 56.25 | 0.3823 |
| Sample 27 | 225 | 112.5 | 12.81 | 0.1708 | |
| XDR | Sample 8 | 450 | 56.25 | 31.25 | 0.5625 |
| Sample 42 | 225 | 250 | 68.75 | 0.5805 | |
| PDR | Sample 1 | 475 | 250 | 56.25 | 0.3434 |
| Sample 3 | 450 | 225 | 100 | 0.6666 | |
Key: MIC = Minimum Inhibitory Concentration, MDR = Multidrug Resistant, XDR = Extensively Drug Resistant, PDR = Pan Drug Resistant, FIC = Fractional Inhibitory Concentration.
Antibiotic resistant bacterial infection is becoming the greatest threat to mankind as mentioned earlier, commercially available antibiotics are being challenged by the pathogen as well. The United States Centers for Disease Control and Prevention estimate the infections caused by antibiotic-resistant bacteria result in some two million cases of illness and 23,000 deaths in the U.S. annually. Additionally, the European Centre for Disease Prevention and Control produces similar numbers, estimating that antibiotic-resistant bacteria kill approximately 25,000 Europeans every year [
This study clearly presents the emerging antibiotic resistant Klebsiella pneumoniae resistant pattern. It is noted that, out of 23 samples 17 of the bacteria were antibiotic resistant which is definitely challenging to medical science. A recent study figured out that a woman died in September, 2016 at Nevada, USA was infected with Klebsiella bacteria which were resistant to 26 different antibiotics. Indeed, the bacteria were resistant to all available antimicrobial drugs in the USA reported by The US Centre for Disease Control and Prevention [
It was found that one of the major factors of increasing antibiotic resistance is the overuse and misuse of antibiotics which is common practice in the countries like India, Pakistan, Bangladesh and Sri Lanka [
Further, antibiotic resistant Klebsiella pneumoniae has been considered as a major threat to global healthcare system. As a result, numerous antibiotic combination studies have been developed against this pathogen. The combination of rifampin and colistin has been found to be bactericidal in KPC-producing K. pneumoniae isolates [
Again, the combination of doripenem and levofloxacin scored 0.5 FIC index against Klebsiella pneumonia infected ICU patient which represents the synergistic effect of these two antibiotics, the study also documented doripenem and colistin as 0.75 FIC index considering additive activity [
This study reveals it’s novelty for combination of ciprofloxacin and chloramphenicol which has not been documented yet. It is the first time when the synergistic activity of these two antibiotics has been found in vitro experiment. The use of antibiotics in combination is already a common hospital procedure in empirical treatment of severe infections but the guideline of using the combination has not been well established. Several investigations have explored the use of various combination regimens for highly antibiotic resistant Klebsiella pneumoniae, but these investigations often lack in vivo validation. It remains unknown which combinations of antimicrobial agents/classes are most effective for the treatment of resistant pathogen [
Another interesting finding is the FIC index of ciprofloxacin and chloramphenicol found in this study is 0.45; compared to literature, the combination of doripenem and levofloxacin scored 0.5, this levofloxacin is from quinolone group other than ciprofloxacin.
In conclusion, it should not be mistaken to assert the upcoming threat of antibiotic-resistant pathogen, particularly Klebsiella pneumonia,that is emerging as a superbug. A developing country like Bangladesh, is going to face a terrible challenge of these emerging pathogen unless and until the frequent misuse and overuse of antibiotic is abridged. Undoubtedly, the healthcare system due to antibiotic resistant bacterial infection also faces economic penalties as well. However, when the question is about life, new methods must be developed to combat these superbugs and existing antibiotic combination can be a good choice. Nevertheless, ciprofloxacin is well established antibiotic having broad spectrum bactericidal activity. Hence, if the efficiency of this antibiotic can be accelerated from combination with other drugs, it might be lifesaving and cost effective as well. Moreover, developing country like ours’ can grab the chance to combat antibiotic resistant Klebsiella pneumoniae from this combination and reduce the mortality rate from prolonged pneumonia since we are endangered floating in the sea of emerging antibiotic resistant pathogen.
Dr. Abu Syeed Mosaddek, professor and head at Pharmacology, Uttara Adhunik Medical College & Hospital, Dr. Most. Fhamida Begum, professor & head at Microbiology, Uttara Adhunik Medical College & Hospital and the authority of National Institute of Diseases of the Chest and Hospital (NIDCH) for giving the opportunity to sample collection.
The authors declare no conflicts of interest regarding the publication of this paper.
Ahmed, A., Aftab, I.B., Mouly, T.F., Mumu, S.K., Mosaddek, A.S.M. and Hossain, M. (2020) A Prospective Study on the Efficiency of Ciprofloxacin in Combination with Chloramphenicol against Multiple Antibiotics Resistant Klebsiella pneumoniae. Advances in Microbiology, 10, 411-421. https://doi.org/10.4236/aim.2020.109030