Effectiveness of selected broad spectrum antibiotics against Pseudomonas species isolated through the use of chloramphenicol

Greenish pigment producing Pseudomonas species were isolated from water samples using nutrient agar supplemented with 50 μg.ml-1 chloramphenicol. Broth cultures of three of the Pseudomonas isolates were prepared for minimum inhibitory concentration (MIC) determination by adjusting their turbidity using sterile normal saline to match the turbidity of McFarland standard No. 1 which is regarded to have a cell density of about 3 × 108 cfu/ml. The MIC of ciprofloxacin, erythromycin, and tetracycline against the three isolates was determined using the agar dilution method. The concentrations of the antibiotics used are 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0, 320.0, and 640.0 μg/ml. The results showed that the MIC of ciprofloxacin against the Pseudomonas isolates ranged from 5 – 640 μg/ml; the MIC of tetracycline against the isolates ranged from 5 – 640 μg/ml; and the MIC of erythromycin against the isolates ranged from 40 80 μg/ml. It is thus concluded that multidrug resistant species of Pseudomonas can be generated as a result of using chloramphenicol for the isolation of Pseudomonas, and that erythromycin can be effective in treating infections caused by Pseudomonas species that are resistant to ciprofloxacin and tetracycline.


Introduction
Cultures of Pseudomonas species are often required for research, environmental and industrial application.This is due to their ability to metabolize and transform several organic compounds, and also their ability to produce biosurfactants and other organic molecules [1][2][3].Due to their potential importance and application, these organisms are often sought after by researchers and bio-industrialists.Isolation of Pseudomonas species from environmental media using nutrient agar supplemented with chloramphenicol has been suggested by Peekate and Abu [4].Their suggestion was based on the observation that certain species of Pseudomonas produces greenish pigment during growth in the presence of chloramphenicol.
Chloramphenicol is a broad spectrum antibiotic used in the treatment of infections such as conjunctivitis, typhoid fever, and otitis media [5][6][7][8].Been a broad spectrum antibiotic, it is expected to be effective in the treatment of infections caused by different bacterial groups.Chloramphenicol is bacteriostatic in nature [9]; it does not actually kill bacterial pathogens, but stops them from multiplying and carrying out certain metabolic activities.As a result of not been killed, some bacterial pathogens develop resistance to the antibiotic, especially when the antibiotic is applied at sub-inhibitory concentrations.
The use of chloramphenicol in the isolation of Pseudomonas, while suppressing the population of other bacteria, can lead to the development of multi-drug resistance in the Pseudomonas species isolated.Determining the potency of selected broad spectrum antibiotics against bacteria isolated using chloramphenicol or other antibiotics is thus necessary so as to be prepared for laboratory acquired infections that may occur due to work with such bacteria.This will also make known the antibiotic of choice for treatment of infections that could arise as a result of escape of such bacteria from the laboratory.
Infections caused by multi-drug resistant species of Pseudomonas can be tackled through the use of broad spectrum antibiotics.Broad spectrum antibiotics, among other usefulness, are useful for treating infections caused by antibioticresistant bacteria [10].Examples of broad spectrum antibiotics include amoxicillin, augmentin, imipenem, chloramphenicol, ciprofloxacin, erythromycin, and tetracycline [9,11].Before the use of a broad spectrum antibiotic in the treatment of infection caused by an antibiotic-resistant bacterium, the effectiveness of the broad spectrum antibiotic against the resistant bacterium should be known so as to aid clinicians in making empirical decisions and prevent the further development and spread of multidrug resistant-bacteria.In investigating the effectiveness of an antimicrobial agent against a bacterial pathogen, the minimum inhibitory concentration (MIC) of the antimicrobial for the pathogen is usually determined through the agar dilution, broth dilution, or disc diffusion method [12][13].
The aim of this study is to determine the effectiveness of selected broad spectrum antibiotics, using agar dilution method, in restricting the growth of Pseudomonas species isolated through the use of agar media supplemented with Chloramphenicol.

Isolation of chloramphenicol resistant Pseudomonas species from water
Water samples were collected from a gutter within the Rivers State University campus, Nigeria, and from a river located near the University.The water samples were collected using 50 ml sterile water sampling bottles.The samples were taken to the Microbiology laboratory of the Rivers State University, and analyzed for total heterotrophic bacterial count using nutrient agar (Titan Biotech Ltd., India), and greenish pigment producing Pseudomonas species using nutrient agar supplemented with 50 μg.ml - chloramphenicol.Inoculated nutrient agar plates were incubated at 37 °C for 24 hours, while the inoculated nutrient agar plates supplemented with chloramphenicol were incubated at ambient temperatures (28 -32 °C) for 48 hours.

Identification of the isolates
Greenish pigment producing colonies that grew on nutrient agar supplemented with 50 μg.ml - chloramphenicol were sub-cultured unto sterile nutrient agar plates to get pure isolates, and then stock culture of the pure isolates were prepared.The isolates were subjected to the Gram staining and microscopic examination, and the following physicochemical/biochemical tests: catalase, oxidase, motility, citrate utilization, indole production, MRVP (Methyl Red-Vogues Proskauer), blood haemolysis, casein hydrolysis, lecithinase production, and fermentation tests using glucose, lactose, maltose, sucrose, mannitol, xylose, starch, and glycerol.

Preparation of selected chloramphenicol resistant Pseudomonas species for MIC determination
Broth cultures of three of the isolates confirmed to belong to Pseudomonas species were prepared by inoculating the isolates into sterile Nutrient broth (Titan Biotech Ltd., India).The inoculated broths were incubated at 37 °C for 24 hours.After 24 hours, the absorbances of the broths were read using a 721 VIS Spectrophotometer (Huanghua Faithful Instrument Co. Ltd, China) set at 600 nm.The turbidity of the broths was then adjusted, using sterile normal saline, to the turbidity of McFarland standard No. 1 which is agreed to portray a broth culture having a cell density of about 3 × 10 8 cfu/ml [14].The absorbance of the McFarland standard at 600 nm as determined using the 721 VIS Spectrophotometer was 0.192.The volume of normal saline added to the broth cultures to achieve the turbidity of McFarland standard was determined using some arithmetic calculations.

Determination of the MIC of selected broad spectrum antibiotics against the selected isolates
The MIC of three broad spectrum antibiotics (ciprofloxacin, erythromycin, and tetracycline) against the three isolates was determined using the agar dilution method [12].Different sets of nutrient agar plates having increasing concentrations of each of the antibiotics were prepared.The concentrations of the antibiotics used include 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0, 320.0, and 640.0 μg/ml.To achieve these concentrations in the nutrient agar plates, stock solutions of 1000 μg/ml of the antibiotics were first prepared.The volume of the antibiotic stock solutions to be added to prepared molten agar medium so as to achieve the different concentrations were determined using equation 1 (Eq.1), adapted from the equation M1V1 = M2V2 [15].
Where VAB is the volume of the antibiotic stock solution required, MAB is the antibiotic concentration specified for the nutrient agar medium, and VMD is the volume of the nutrient agar medium to be prepared.
On preparation of the nutrient agar media, after sterilization, the media were allowed to cool to about 50 °C before addition of the calculated volume of the antibiotic stock solution.The media now containing the antibiotics were poured into appropriately labeled sterile Petri dishes, allowed to solidify, and then dried in a size one hot box oven (Gallenkamp and Co. Ltd., England) set at 50 °C.Broth cultures (0.1 ml) of the isolates adjusted to the McFarland No. 1 standard were inoculated onto their respective plates containing the antibiotics, and incubated at 37°C for 24 hours.After incubation, the plates were checked for the presence or absence of growth.The presence or absence of growth in each set of plates was then used to adjudge the MIC of the antibiotics against the isolates.

Bacterial populations of the water samples
The total heterotrophic bacterial population (THB) and the greenish pigment producing bacterial population of water samples from the gutter and the river is presented in Table 1.From the Table it can be seen that the greenish pigment producing bacterial population of the samples are lower than the total heterotrophic bacterial population.THB -total heterotrophic bacterial population, GPB -greenish pigment producing bacterial population, GT -gutter water, RW -river water Selected greenish pigment producing the isolates were coded as follows; CH1, CH2, CH3, and CH4.Results generated from microscopic examination of their stained cells, and the physicochemical/biochemical tests carried out on them is presented in Table 2. On scrutiny of Table 2, it can be seen that the isolates displayed almost similar results for the different physicochemical/biochemical tests used.Determination of the MIC of the antibiotics, ciprofloxacin, erythromycin, and tetracycline, against the isolates CH1, CH3, and CH4 is presented in Table 3 -5.From the Tables it can be seen that the MIC of ciprofloxacin against isolate CH1, CH3, and CH4 is 5 μg/ml, 640 μg/ml, and 5 μg/ml respectively; the MIC of tetracycline against the isolates is 20 μg/ml, 640 μg/ml, and 5 μg/ml respectively; the MIC of erythromycin against the isolates is 40 μg/ml, 80 μg/ml, and 80 μg/ml respectively.It can thus be deduced that isolate CH3 was resistant to ciprofloxacin and tetracycline, while isolate CH1 and CH4 were fairly susceptible to the three antibiotics.

Discussion
Culturing environmental or clinical samples on agar media supplemented with antibiotics for the isolation of greenish pigment producing Pseudomonas species can lead to the emergence of multidrug resistant Pseudomonas species.Infections caused by multidrug resistant species of Pseudomonas can be tackled through the use of broad spectrum antibiotics.
In this research, the effectiveness of three broad spectrum antibiotics (ciprofloxacin, tetracycline, and erythromycin) against Pseudomonas species isolated through the use of nutrient agar supplemented with 50 μg/ml chloramphenicol was investigated.Colonies of Pseudomonas species that grew on the media were initially identified by the green pigment they produced which diffused into the media.The similar results displayed by the isolates are similar to the results usually displayed by species of Pseudomonas to the physicochemical/biochemical tests used.This conclusion was drawn by comparison with information in Prescott et al. [9], Lysenko [16], and Reynolds et al. [17].
The MIC of ciprofloxacin, tetracycline, and erythromycin against three of the green pigment producing Pseudomonas isolates were determined so as to know the effectiveness of the antibiotics in treating infections that may be caused by antibiotic-resistant Pseudomonas species.From the results obtained it can be deduced that one of the Pseudomonas isolate was highly resistant to ciprofloxacin and tetracycline; the MIC of these antibiotics against the isolate was as high as 640 μg/ml.This is in agreement with the work of Hamud-Socoro [18], in which it was shown that a species of Pseudomonas (P.aeruginosa) became resistant to tetracycline after been grown in a medium containing low concentration of the antibiotic.In another related work, it was shown that a strain of P. aeruginosa has intrinsic resistance to tetracycline and some other antibiotics [19].However, strains of another species of Pseudomonas (P.fluorescens) have been shown to be highly susceptible to tetracycline at a MIC of 0.305 μg/ml [20].The strains were variants resistant to penicillin G, streptomycin, lincomycin and rifampicin which were developed from a colistinsensitive isolate of P. fluorescens LRC-R73.In this work one of the Pseudomonas isolate was susceptible to tetracycline at a MIC of 5 μg/ml, and another at a MIC of 20 μg/ml.Due to the differences in response to tetracycline, it can be concluded that the three Pseudomonas isolates belong to different species.
Two of the Pseudomonas isolates were susceptible to ciprofloxacin at a MIC of 5 μg/ml, while one of the isolate was highly resistant to the antibiotic.A species of Pseudomonas (P.aeruginosa) have been shown to become resistant to ciprofloxacin after exposure to the antibiotic [21].However, only a small population of the Pseudomonas species survived treatment with the antibiotic, and became resistant to it.Pseudomonas species, especially P. aeruginosa, are usually fully susceptible to ciprofloxacin [22].Resistance to ciprofloxacin occurs due to mutation in certain genes, and at a slow rate.Also only a small fraction of the population may become resistant after exposure.As seen in this study only one of the Pseudomonas isolates was resistant to ciprofloxacin.
The three Pseudomonas isolates were susceptible to erythromycin at MIC values ranging from 40 -80 μg/ml.Not much work on the activity of erythromycin on Pseudomonas species can be found in online libraries.However, in a review carried out by Morita et al. [23], P. aeruginosa cells are said to be intrinsically resistant to macrolides, and that the MIC of erythromycin for a strain of P. aeruginosa is about 512 μg/ml.The variance observed between the MIC in the review and this work could be attributed to the methods, media, quantity of cells used, and strain of the organisms investigated.For instance, in the review the MIC was cited for test carried out using Mueller-Hinton broth, whereas in this work the MIC was determined using an all purpose medium, and a solid medium not a liquid medium (broth).
The confirmation or disapproval of the effectiveness of erythromycin against resistant Pseudomonas species can be achieved by further research in this area.Also, future work can be carried out to determine the effectiveness of erythromycin against resistant isolates of other bacterial genus.However, for the time been, it has been shown in this work that erythromycin is effective against Pseudomonas species that are resistant to ciprofloxacin and tetracycline.

Conclusion
Multidrug resistant species of Pseudomonas can be generated in the laboratories as a result of using chloramphenicol for the isolation of Pseudomonas.Escape of these organisms from Microbiology laboratories can lead to the emergence and spread of infections that could be difficult to treat.More often, infections caused by multidrug resistant bacteria are treated with the use of broad spectrum antibiotics.It thus becomes important to know which broad spectrum antibiotic will actually work in such case scenario.In this work it has been shown that erythromycin is effective against Pseudomonas species that are resistant to ciprofloxacin and tetracycline as a result of growth on medium containing chloramphenicol.This implies that erythromycin could be used in the treatment of infections caused by multidrug resistant Pseudomonas species.

Disclosure of conflict of interest
There are no conflicts of interest.

Table 1Bacterial
population of the sampled water bodies

Table 2
Identification test results of selected greenish pigment producing bacterial isolates β-H -beta haemolysis, A -only acid produced, 0 -no change.

Table 4
MIC determination of tetracycline against the isolates

Table 5
MIC determination of Erythromycin against the isolates