COMPARATIVE INVITRO ACTIVITY OF GEMIFLOXACIN AND SOME OTHER QUINOLONES
AGAINST SOME CLINICAL BACTERIAL ISOLATES
Ching, Fidelis Poh*1, Josephs, Godwin Chimechefulam2 and Ossai, Awele Maureen2
*1
Department of Pharmacology, Faculty of Basic Medical Sciences, College of Health Sciences,
Niger Delta University, Wilberforce Island, Yenagoa, Bayelsa State,
Nigeria. E-mail:
fidelching@yahoo.ca
Josephs, Godwin Chimechefulam
2Department
of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Benin, Benin
City, Edo State, Nigeria E-mail:godwinjosephs@yahoo.com
*Corresponding author:
Dr Ching, Fidelis Poh
Department of Pharmacology, Faculty of Basic Medical Sciences, College of Health Sciences,
Niger Delta University, Wilberforce Island, Yenagoa, Bayelsa State,
Nigeria. Phone:
+2348067541738, E-mail: fidelching@yahoo.ca
COMPARATIVE INVITRO ACTIVITY OF GEMIFLOXACIN AND SOME OTHER QUINOLONES
AGAINST SOME CLINICAL BACTERIAL ISOLATES
ABSTRACT
Background: Resistance to antibacterial agents has become a global problem and has made
many existing antibiotics less effective in the treatment of infections for which they were
effective. Gemifloxacin is a third generation fluoroquinolone with broad spectrum antimicrobial
and enhanced activity against both aerobic and anaerobic Gram – positive bacteria. It is
recommended for the treatment of respiratory tract infections.
Objectives: This study evaluated and compared with ciprofloxacin, ofloxacin and norfloxacin its
in vitro antibacterial activity against Staphylococcus aureus, Escherichia Coli, Alcaligenes
faecalis, Pseudomonas aeruginosa, Serratia marcescens, Enterobacter aerogenes, Proteus
vulgaris and Klebseilla pneumoniae.
Methods: The minimum inhibition concentration (MIC) and zones of inhibition assays were
employed. In the MIC assays final nutrient agar extracts concentrations of 5, 7.5, 10, 15, 20, 25
and 30µg/ml of gemifloxacin, ciprofloxacin, ofloxacin, and norfloxacin were used. In the zones
of inhibition assay 0.2ml of the MIC of gemifloxacin against each bacterium was used.
Results: The MICs of gemifloxacin were 7.5µg/ml for Proteus vulgaris, 10µg/ml for Staph.
aureus and Enterobacter aerogenes, 15µg/ml for E. coli, 20µg/ml for Pseudomonas aeruginosa,
25µg/ml for Klebsiella pneumonia and 30µg/ml for Alcaligenes faecalis and Serratia
marcescens. The clinical isolates were most sensitive to ofloxacin with MICs of 7.5µg/ml for
Proteus vulgaris and 10µg/ml for the other isolates. The isolates were least susceptible to
norfloxacin with MICs of 10µg/ml for Proteus vulgaris and 30µg/ml for the other isolates.
The results show that the clinical isolates were most sensitive to ofloxacin followed by
gemifloxacin, ciprofloxacin and norfloxacin respectively.
Conclusion: The study has shown that gemifloxacin possess antibacterial activity against the
pathogenic bacteria and could have a place in the treatment of the infections caused by the
organisms.
Keywords:
Gemifloxacin,
urinary
tract
infections,
invitro
activity.
INTRODUCTION
The quinolones are synthetic broad spectrum antibiotics. The first quinolone, nalidixic acid was
introduced essentially for the treatment of urinary tract infections. The newer quinolones, the
fluoroquinolones, however, are used for urinary and respiratory tracts infections as well as
other infections.1 The second generation quinolones which include ciprofloxacin, ofloxacin, and
norfloxacin are used essentially for the treatment of urinary tract infections while the third
generation such as gemifloxacin, levofloxacin and moxifloxacin are used essentially for the
treatment of respiratory tract infections.2 Ciprofloxacin is also used for the treatment of other
infections such as bones and joints, endocarditis, gastroenteritis, malignant otitis externa, lower
respiratory tract infections, chronic bacterial prostatitis, anthrax, acute sinusitis although not as
first line.3 Ofloxacin is also used for the treatment of acute bacterial exacerbation of chronic
bronchitis, community acquired pneumonia, uncomplicated skin and skin structure infections,
non-gonococcal urethritis and cervicitis, acute pelvic inflammatory disease and prostatitis. 4 In
adult population, norfloxacin is used for the treatment of uncomplicated and complicated
urinary tract infections.5 Gemifloxacin is effective in the treatment of penicillin-sensitive and
penicillin-resistant Streptococcus pneumoniae and atypical pathogens which include
mycoplasma pneumoniae and chlamydia pneumoniae.1 The development of resistance to
existing antimicrobial agents has become a global concern. The treatment of urinary tract
infections with the traditionally used quinolones is being threatened by the emergence of
multidrug resistant pathogens.2,5,6 This emergence of the multidrug resistance pathogens
necessitates the need to explore the other quinolones with broad spectrum activity in the
treatment of urinary tract infections and other infections. Gemifloxacin which has shown
potent activity against penicillin, macrolide and other quinolone resistant bacteria.6,7,8 could be
of great importance in the treatment of urinary tract infections and other infections in this era
of increasing antimicrobial resistance.
This study evaluated the in vitro activity of gemifloxacin against clinical bacterial isolates and
compared the activity with that of ciprofloxacin, ofloxacin and norfloxacin against the same
isolates.
MATERIALS AND METHODS
Drugs and Chemicals
The pure drugs which included gemifloxacin, ciprofloxacin, ofloxacin and norfloxacin were
products of Sigma Aldrich Laborchemikallien, GmBH, Germany. They were in safe conditions for
use whiles the microbiological media, nutrient agar, nutrient both and MacConkey agars were
also products of Sigma Aldrich Laborchemikallien, GmBH, Germany.
Clinical Bacterial Isolates
Eight pathogenic bacteria were used. These included Staphylococcus aureus (Staph. aureus),
Escherichia Coli (E. coli), Alcaligenes faecalis (A. faecalis), Pseudomonas aeruginosa (P.
aeruginosa), Serratia marcescens (S. marcescens), Enterobacter aerogenes (E. aerogenes),
Proteus vulgaris (P. vulgaris) and Klebseilla pneumoniae (K. pneumoniae). They were obtained
from the University of Benin Teaching Hospital Medical Microbiology Laboratory stock Unit.
They had been isolated from samples of patients for clinical diagnosis of their infections. They
were authenticated using standard morphological and biochemical assays.9 in the Department
of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Benin. The bacterial
isolates were maintained in nutrient agar slants and sub cultured at regular intervals to ensure
their viability and purity.
Preparation of Concentrations of drugs for Minimum Inhibitory Concentration (MIC) assays.
From a stock solution of 1000 µg/ml of each of the drugs final concentrations of 5, 7.5, 10,15,
20, 25, and 30µg/ml in equivalent volumes of 0.1 ml, 0.15ml, 0.2ml, 0.3ml, 0.4, 0.5ml and 0.6ml
respectively made up to 20 ml with nutrient agar were used for antibacterial assay.
Preparation of inoculums
The respective bacterial isolates from the nutrient agar slants were sub cultured in sterile
nutrient broth and incubated at 370C overnight. The overnight nutrient broth culture was
diluted serially to obtain 106CFU/ml and used after every fresh preparation.
ANTIBACTERIAL ASSAY
Minimum Inhibitory Concentrations (MIC) of the drugs
The agar dilution method described by George and Robert was used.10 The minimum inhibitory
concentrations of the drugs were evaluated using final concentrations of 5, 7.5, 10, 15, 20 and
30 mg/ml in equivalent volumes of 0.1ml, 0.15ml, 0.2ml, 0.3ml, 0.4, 0.5 and 0.6 ml respectively
made up to 20 ml with nutrient agar.
The minimum inhibitory concentrations were
determined against Staphylococcus aureus, Escherichia Coli, Alcaligenes faecalis, Serratia
marcescens, Enterobacter aerogenes, Klebseilla pneumoniae, Pseudomonas aeruginosa and
Proteus vulgaris. Each concentration of each drug was in triplicate plates which were labelled
accordingly with the clinical bacterial isolate. Each plate was inoculated with 0.1ml of 10 6
CFU/ml of overnight nutrient broth culture of the clinical bacterial isolate. The inoculums were
spread uniformly on the surface of the agar with the aid of a sterile glass spreading rod. The
plates were incubated at 37 oC and observed for growth after 24 hours.
Evaluation of gemifloxacin antibacterial activity against the Clinical Isolates using Zones of
Inhibition
The agar well diffusion method as described by Perez et al. was used.11 The minimum
inhibitory concentration (MIC) against each bacterium exhibited by gemifloxacin was used in
the zones of inhibition assay. The zones of inhibition assay were evaluated against
Staphylococcus aureus, Escherichia Coli, Alcaligenes faecalis, Serratia marcescens, Enterobacter
aerogenes, Klebseilla pneumoniae, Pseudomonas aeruginosa and Proteus vulgaris. Petri dishes
in triplicates were labelled with each of the clinical bacterial isolates and 20ml of nutrient agar
pipetted into them aseptically. The nutrient agar was allowed to set and was inoculated with
0.1ml of 106 CFU/ml of an overnight culture of each of the clinical isolates, in separate petri
dishes. The inoculums were spread uniformly on the surface of the agar with the aid of a
sterile glass spreading rod. Using a sterile cork borer, wells of 6mm in diameter were made in
the nutrient agar of each plate and labelled accordingly. In each of the wells, 0.2ml of each of
gemifloxacin minimum inhibitory concentration against each bacterium was aseptically
pipetted and labelled accordingly. The plates were left for a while for the drug to diffuse into
the agar. The plates were incubated at 37 oC for 24 hours and examined for zones of inhibition
around the wells. The zones of inhibition were measured and recorded.
The mean and
standard error of mean (Mean ± SEM) was determined for the triplicate plates in each case.
RESULTS
The results of the minimum inhibitory concentration assays of gemifloxacin, ciprofloxacin,
ofloxacin and norfloxacin are presented in Tables 1, 2, 3 and 4. The MICs of gemifloxacin were
7.5µg/ml for Proteus vulgaris, 10µg/ml for Staph. aureus and Enterobacter aerogenes, 15µg/ml
for E. coli, 20µg/ml for Pseudomonas aeruginosa, 25µg/ml for Klebsiella pneumonia and
30µg/ml for Alcaligenes faecalis and Serratia marcescens (Table 1).
Table 1: Minimum inhibitory concentration (MIC) of gemifloxacin against the clinical bacteria
isolates
Clinical bacteria isolates
Staphylococcus aureus
Final concentrations in nutrient agar (µg/ml)
5.0
7.5
10.0
15.0 20.0
25.0
30.0
++
+
-
Escherichia coli
++
++
+
-
-
-
-
Pseudomonas aeruginosa
++
+
+
+
-
-
-
Enterobacter aerogenes
+++
++
-
-
-
-
-
+
-
-
-
-
-
-
Alcaligenes faecalis
+++
++
++
+
+
+
-
Serratia marcescens
+++
++
++
+
+
+
-
Klebsiella pneumonia
++
++
+
+
+
-
-
Proteus vulgaris
+++, heavy growth of colonies, ++, moderate growth of colonies, +, minimal growth and -, no
growth of colonies
The MICs of ciprofloxacin were 7.5µg/ml for Proteus vulgaris, 10µg/ml for Staph. aureus,
25µg/ml for E. coli and Pseudomonas aeruginosa, 30µg/ml for Enterobacter aerogenes,
Klebsiella pneumonia, Alcaligenes faecalis and Serratia marcescens (Table 2).
Table 2: Minimum inhibitory concentration (MIC) of ciprofloxacin against the clinical bacteria
isolates
Clinical bacteria isolates
Staphylococcus aureus
Final concentrations in nutrient agar (µg/ml)
5.0
7.5
10.0
15.0 20.0
25.0
30.0
++
+
-
Escherichia coli
+++
++
++
+
+
-
-
Pseudomonas aeruginosa
+++
++
++
+
+
-
-
Enterobacter aerogenes
+++
++
++
++
+
+
-
+
-
-
-
-
-
-
Alcaligenes faecalis
+++
+++
+++
++
++
+
-
Serratia marcescens
+++
+++
++
++
++
+
-
Klebsiella pneumonia
+++
+++
++
++
++
+
-
Proteus vulgaris
+++, heavy growth of colonies, ++, moderate growth of colonies, +, minimal growth and -, no
growth of colonies
The MICs of ofloxacin were 7.5µg/ml for Pseudomonas aeruginosa and Proteus vulgaris,
10µg/ml for Staph. aureus, E. coli, Enterobacter aerogenes, Klebsiella pneumonia, Alcaligenes
faecalis and Serratia marcescens (Table 3).
Table 3: Minimum inhibitory concentration (MIC) of ofloxacin against the clinical bacteria
isolates
Clinical bacteria isolates
Staphylococcus aureus
Final concentrations in nutrient agar (µg/ml)
5.0
7.5
10.0
15.0 20.0
25.0
30.0
++
+
-
Escherichia coli
++
+
-
-
-
-
-
Pseudomonas aeruginosa
++
-
-
-
-
-
-
Enterobacter aerogenes
++
+
-
-
-
-
-
Proteus vulgaris
+
-
-
-
-
-
-
Alcaligenes faecalis
++
+
-
-
-
-
-
Serratia marcescens
++
+
-
-
-
-
-
Klebsiella pneumonia
++
+
-
-
-
-
-
++, moderate growth of colonies, +, minimal growth and -, no growth of colonies
The MICs of norfloxacin were 10µg/ml for Proteus vulgaris, 30µg/ml for Staph. aureus, E. coli,
Enterobacter aerogenes, Klebsiella pneumonia, Alcaligenes faecalis, Pseudomonas aeruginosa
and Serratia marcescens (Table 4).
Table 4: Minimum inhibitory concentration (MIC) of norfloxacin against the clinical bacteria
isolates
Clinical bacteria isolates
Staphylococcus aureus
Final concentrations in nutrient agar (µg/ml)
5.0
7.5
10.0
15.0 20.0
25.0
30.0
+++
++
++
++
++
+
-
Escherichia coli
+++
+++
++
++
++
++
-
Pseudomonas aeruginosa
+++
+++
++
++
++
++
-
Enterobacter aerogenes
+++
+++
+++
+++
++
++
-
Proteus vulgaris
++
+
-
-
-
-
-
Alcaligenes faecalis
+++
+++
+++
++
++
++
-
Serratia marcescens
+++
+++
+++
++
++
++
-
Klebsiella pneumonia
+++
+++
+++
++
++
++
-
+++, heavy growth of colonies, ++, moderate growth of colonies, +, minimal growth and -, no
growth of colonies
In Table 5 is presented the MICs of gemifloxacin in comparison to ciprofloxacin, ofloxacin and
norfloxacin. The results show that the clinical isolates were most sensitive to ofloxacin followed
by gemifloxacin, ciprofloxacin and norfloxacin respectively (Table 5).
Table 5: Minimum inhibitory concentration (MIC) values of gemifloxacin, ciprofloxacin,
ofloxacin and norfloxacin against the clinical bacteria isolates
Clinical bacteria isolates
Staphylococcus aureus
Minimum inhibitory concentration values (µg/ml)
GEM
CIP
OFL
NOR
10
10
10
30
Escherichia coli
15
25
10
30
Pseudomonas aeruginosa
20
25
10
30
Enterobacter aerogenes
10
30
7.5
30
Proteus vulgaris
7.5
7.5
10
10
Alcaligenes faecalis
30
30
7.5
30
Serratia marcescens
30
30
10
30
Klebsiella pneumonia
25
30
10
30
The zones of inhibition for gemifloxacin are show in Table 6. The zones of inhibition range from
17.0 ± 0.11mm to 42.0 ± 0.15mm for Enterobacter aerogenes and Serratia marcescens
respectively (Table 6).
Table 6: Antibacterial activity of gemifloxacin against clinical bacterial
isolates using zones of inhibition
Clinical bacteria isolates
Staphylococcus aureus
Zone of inhibition (mm)
17.5 ± 0.22
Escherichia coli
18.0 ± 0.30
Pseudomonas aeruginosa
32.5 ± 0.14
Enterobacter aerogenes
17.0 ± 0.11
Proteus vulgaris
25.0 ± 0.21
Alcaligenes faecalis
23.5 ± 0.23
Serratia marcescens
42.0 ± 0.15
Klebsiella pneumonia
37.0 ± 0.41
Zones of inhibition are Mean + SEM of measurements for triplicate plates.
DISCUSSION
The emergence of antibiotic resistance is on the increase and has become a global concern.12
Various efforts are being made to combat and cope with this menace which poses a serious
problem to antimicrobial chemotherapy success. Current efforts are focused on the
improvement of antimicrobial spectra of most antimicrobial agents. However, despite their
improved activities, newer drugs still carry the risk of resistance selection, particularly
Staphylococcus pathogens that have already intermediate resistance to antimicrobials.13 This is
a clinical problem, especially with methicillin resistant Staphylococcus aureus (MRSA) isolates
which are widely resistant to quinolones.13 Another possible approach to the problem could be
to explore existing antimicrobial agents with broad spectra of activity for possible activity in
infections for which they are not traditional indicated. Our study evaluated and compared the
in-vitro antibacterial activity of gemifloxacin, a respiratory quinolone and other quinolones
against clinical bacterial isolates of other infections. The bacterial isolates included
Staphylococcus aureus, Escherichia coli, Alcaligenes faecalis, Serratia marcescens, Enterobacter
aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus vulgaris. The results
indicated marked invitro antibacterial activity against the isolates in the minimum inhibitory
concentration assays. The MICs of the drugs evaluated ranged from 7.5µg/ml to 30µg/ml. The
isolates were most sensitive to ofloxacin, a urinary tract infection quinolone, with a maximum
MIC of 10µg/ml for most of the isolates tested followed by gemifloxacin. The MIC assays
indicate that the isolates were least sensitive to norfloxacin with MIC of 30µg/ml for all the
isolates compared to gemifloxacin and the other quinolones. The minimum inhibitory
concentrations are important to confirm the resistance of microorganisms to an antimicrobial
agent and also to monitor the activity of the new antimicrobial agents.14 That the drugs
evaluated produced remarkable growth inhibitions at these concentrations suggest that they
could be good candidates for infections against these isolates. Gemifloxacin in the zones of
inhibition assays produced zones of inhibition for all the isolates tested. The largest zone of
inhibition was observed for Serratia marcescens and the least for Enterobacter aerogenes.
None of the isolates exhibited resistance to gemifloxacin. The zones of inhibition indicate the
degree of susceptibility to the drugs. Gemifloxacin may have a place in the future, as an
antibacterial agent in the treatment of infections caused by some of the isolates tested as is
currently recommended mostly for respiratory tract infections. It can be concluded that the
drugs evaluated in this study could be suitable candidates for treatment of infections caused by
the isolates tested. However in vivo studies are required to ascertain the pharmacokinetics and
Pharmacodynamics of the drugs in disease state.
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