Saudi Journal of Biological Sciences (2013) 20, 7983

King Saud University

Saudi Journal of Biological Sciences

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ORIGINAL ARTICLE

In vitro trials of some antimicrobial combinations against

Staphylococcus aureus and Pseudomonas aeruginosa
Zafar Ahmed *, Shaukat Saeed Khan, Mahnaaz Khan
Department of Microbiology, Saia P.G. College of Science and Education, Bhopal 462001, India
Received 19 June 2012; revised 13 October 2012; accepted 14 October 2012
Available online 10 November 2012

KEYWORDS
Antimicrobial;
Synergism;
S. aureus;
P. aeruginosa

Abstract Staphylococcus aureus and Pseudomonas aeruginosa are rapidly increasing as multidrug
resistant strains worldwide. In nosocomial settings because of heavy exposure of different antimicrobials, resistance in these pathogens turned into a grave issue in both developed and developing
countries. The aim of this study was to investigate in vitro antibiotic synergism of combinations of
b-lactamb-lactam and b-lactamaminoglycoside against clinical isolates of S. aureus and
P. aeruginosa. Synergy was determined by checkerboard double dilution method. The combination
of amoxicillin and cefadroxil was found to be synergistic against 47 S. aureus isolates, in the FICI
range of 0.140.50 (81.03%) followed by the combination of streptomycin and cefadroxil synergistic
against 44 S. aureus isolates in the FICI range of 0.030.50 (75.86%). The combination of streptomycin and cefadroxil was observed to be synergistic against 39 P. aeruginosa isolates in the FICI range of
0.160.50 (81.28%). Further actions are needed to characterize the possible interaction mechanism
between these antibiotics. Moreover, the combination of streptomycin and cefadroxil may lead to
the development of a new and vital antimicrobial against simultaneous infections of S. aureus and
P. aeruginosa.
2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: MIC, minimum inhibitory concentration; FICI,

fractional inhibitory concentration index; MRSA, methicillin resistant
Staphylococcus aureus.
* Corresponding author. Address: Flat No. 2, Mahaveer stores,
Opp. Popular Prestige, Survey 1, Warje Gaon, Warje, Pune 411058,
India. Tel.: +91 86 00107925; fax: +91 20 66754226.
E-mail address: zafar_2085@[Link] (Z. Ahmed).
Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

The current clinical scenario indicates that millions of people

are becoming infected with nosocomial infection leading to
numerous deaths annually worldwide and to heavy economic
burden in both developed and developing countries (WHO,
2002). Emerging trends in nosocomial infections signal high
alerts towards multidrug resistant pathogens. Studies show
that 70% of nosocomial infections are due to antibiotic
resistant strains (Burke, 2003; Safdar et al., 2001). Major
causative agents include antibiotic resistant strains of
Staphylococcus aureus and Pseudomonas aeruginosa. Both of
these organisms are among the most common isolates from
burnt patients especially under nosocomial conditions (Haghi
et al., 2010).

1319-562X 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
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0
0
0
27.08
12.50
16.67
10.42
18.75
0
47.92
39.58
2.08
14.58
29.17
81.28
(a) SN: synergy, (b) PS: partial synergy, (c) AD: additive, (d) IN: indifference, (e) AN: ANTAGONISM.

8.62
41.38
10.34
5.17
18.97
1.72
5.17
22.41
12.07
81.03
17.24
75.86
-

AN (%)
IN (%)
AD (%)

0
0
0
-

Amoxicillin
Ampicillin
Streptomycin
Cefadroxil

0 (0)
0 (0)
22.92 (11)
29.17 (14)

AN (%)
IN (%)
AD (%)
PS (%)
SN (%)

Antibiotic in combination with Cefadroxil against

P. aeruginosa (n = 48)

Sensitivity
obtained with
antibiotic alone
against P. aeruginosa
(no. of isolates) (%)
Antibiotic
tested

13.79 (8)
8.62 (5)
36.21 (21)
32.76 (19)

Antibiotics susceptibility test was done for all isolates of S.

aureus and P. aeruginosa, using Kirby-Bauer disc diffusion
method (Bauer et al., 1966). Antibiotic discs were obtained
from Himedia Laboratories Pvt. Ltd., Mumbai including
amoxicillin (10 lg), ampicillin (10 lg), streptomycin (10 lg)
and cefadroxil (30 lg). The diameters of the zones of inhibition
were interpreted in Tables 2A through 2I (Zone Diameter
Interpretative Standards and equivalent Minimum Inhibitory
Concentration Breakpoints) of the NCCLS M100-S12

Amoxicillin
Ampicillin
Streptomycin
Cefadroxil

2.3. Susceptibility test

PS (%)

Antimicrobial agents, namely amoxicillin and streptomycin

were procured from Himedia Laboratories Pvt. Ltd., Mumbai,
India, cefadroxil was procured from IFPRESS, Mumbai, India
and ampicillin (Roscillin) was procured from Ranbaxy. All
drugs were dissolved in their respective solvents and diluted
in deionized water and ltered through 0.22 l Millipore lter.
Drug stocks were stored at 20 C.

SN (%)

2.2. Antimicrobial agents

Antibiotic in combination with cefadroxil against

S. aureus (n = 58)

A total of 58 S. aureus and 48 P. aeruginosa isolates included in

the present study were taken from the collection of the Vishakha Clinical Microbiology Laboratory, Nagpur, India. The
clinical isolates were from different specimens like pus, swab,
sputum, blood and wound discharge of patients attending
the health centres of Nagpur district. Their species identity
was conrmed by biochemical tests including catalase, coagulase, and by growth characteristics on Mannitol salt agar for S.
aureus, and Glucose utilization, Indole, MR (Methyl red), VP
(Voges-Proskauer), Catalase, Oxidase, and by growth characteristics on Cetrimide agar for P. aeruginosa.

Sensitivity
obtained
with antibiotic
alone against
S. aureus (n) (%)

2.1. S. aureus and P. aeruginosa isolates

Antibiotic
tested

2. Material and methods

Table 1

In an increasing number of microbial infections of man,

treatment with single antibiotic fails to cure. Some of these
infections, however, respond favourably to combined treatment with two antibiotic drugs. Certain pairs of these drugs
have proved successful in a limited number of clinical
situations. The clinical signicance of in vitro synergism has
not yet been dened in the treatment of many infections. Therefore, this study was undertaken to investigate the effectiveness
of combinations of antibiotics against clinical isolates of S. aureus and P. aeruginosa which are synergistic in vitro. It will be
shown that the use of two antibiotics that are synergistic
in vitro against the microorganism responsible for the infection
may be associated with a signicantly better outcome than that
achieved with a combination which is not synergistic for the
offending microorganism. Synergistic activity of antimicrobials
cannot be assumed and should be tested for, prior to commencing treatment with a combination regimen (Prinsloo et al.,
2008). Therefore this study was aimed to assess the in vitro synergy of three antibiotics against 58 clinical isolates of S. aureus
and 48 clinical isolates of P. aeruginosa. The manuscript does
not contain experiments using animals and human studies.

Z. Ahmed et al.

In vitro interaction of cefadroxil with amoxicillin, ampicillin and streptomycin against S. aureus and P. aeruginosa isolates.

80

In vitro trials of some antimicrobial combinations against Staphylococcus aureus and Pseudomonas aeruginosa

81

(2002), organisms were reported as susceptible, intermediate or

resistant to the agents that have been tested.

In vitro checkerboard studies on the activity of antibiotics

alone and in combination were performed in Muller Hinton
broth (Himedia Ltd.) in tube dilution. Twofold dilutions
(0.125256 lg ml 1) of each drug or drug combination were
tested in two rows. One row was inoculated with 105 organisms/ml of the test organism and the second row was inoculated with the control organism. Results were read after
tubes were incubated at 37 2 C for 18 h. MIC (Minimum
inhibitory concentration) is determined as the lowest concentration of the drugs (alone or in combination) that inhibit
growth. The fractional inhibitory concentration index (FICI)
is dened as the sum of the MIC of each drug when used in
combination divided by the MIC of the drug used alone.
Synergistic effect was recorded when FIC indexes 60.5; partial
synergy when FIC >0.5 but <1.0; additive when FIC = 1.0;
indifferent when FIC >1.0 but <4.0 and antagonistic when
FIC P4.0 (Cai et al., 2007).
3. Results

No. of isolates

The in vitro results of interaction of cefadroxil with amoxicillin, ampicillin and streptomycin are presented in Table 1. Disc
diffusion test results showed that about 90% S. aureus isolates
were resistant to amoxicillin and ampicillin, however both of
these antibiotics were found to be ineffective against all
P. aeruginosa isolates (100% resistance). About 33% and
36% S. aureus isolates and about 29% and 23% P. aeruginosa
isolates were susceptible to cefadroxil and streptomycin
respectively.
The results of checkerboard analysis illustrated (Table 1)
that the combination of cefadroxil and amoxicillin was most
effective against S. aureus isolates with 81.03% (47) synergism
(FIC index 0.140.50) and less than 10% of other effects. FIC
value distribution among 58 S. aureus isolates is presented in
Fig. 1. The same combination of antibiotics found to be marginally synergistic i.e. 14.58% (7) (FIC index 0.090.38) but

Figure 1
isolates.

FICI of antibiotic combinations against S. aureus

No. of isolates

2.4. MIC and FICI determination

Figure 2
isolates.

FICI of antibiotic combinations against P. aeruginosa

higher side towards partial synergism 47.92% (23) (FIC index

0.520.75) and indifference 27.08% (13) (FIC index 1.062.06),
is presented in Fig. 2.
FIC index interpretation for activities of cefadroxil with
ampicillin against S. aureus isolates predominantly showed
indifference 41.38% (24) (FIC index 1.032.5) and same combination of antibiotics against P. aeruginosa isolates predominantly showed partial synergism 39.58% (19) (FIC index 0.52
0.75).
Cefadroxil combined with streptomycin produced predominant synergism 75.86% (44) (FIC index 0.030.50) against S.
aureus isolates and maximum synergism i.e. 81.28% (39) (FIC
index 0.160.50) against P. aeruginosa isolates with least other
effects.
No antagonism with any combination of antibiotics was
observed in the present study.
4. Discussion
Infections caused by S. aureus and P. aeruginosa are increasing
both in hospitals and in general community (Hauser and Sriram, 2005; Maltezou and Giamarellou, 2006). The efcacy of
many antibiotics for treatment of severe infections has become
quite limited due to the development of resistance. The results
from this in vitro study with 58 S. aureus clinical isolates for
their susceptibility against different antibiotics indicated
8.62% and 13.79% sensitivity to ampicillin and amoxicillin
respectively. Cefadroxil and streptomycin were found to be
effective against 32.76% and 36.21% isolates. Our results also
substantiate previous study of Adegoke and Komolafe (2009).
Penicillins and Cephalosporins are the b-lactam antibiotics
which inhibit cell wall synthesis. Resistance against these antibiotics revealed that the resistance is purely plasmid based
since b-lactamase production is plasmid based (Rigby, 1986).
Kondo et al. (1991) had reported good activities of streptomycin (MIC 1.566.25 lg/ml) against all tested strains of MRSA.
The present study showed that 62.07% strains of S. aureus
were resistant to streptomycin.
The in vitro study with 48 clinical isolates of P. aeruginosa
exhibited various degrees of inhibition against different antibiotics. Total 100% resistance was observed against amoxicillin

82
and ampicillin. Whereas, 70.83% and 77.08% resistance was observed against cefadroxil and streptomycin respectively, earlier
studies also showed similar ndings (Anjum and Mir, 2010).
The multidrug resistance among P. aeruginosa isolates may involve reduced cell wall permeability, production of chromosomal and plasmid mediated b-lactamases (Livermore, 1989),
aminoglycoside-modifying enzymes (Livermore, 1987), and an
active multidrug efux mechanism (Shahid and Malik, 2004).
The rational for combination therapy is essential to reduce
the chances of selection of resistant mutants during therapy, as
well as to exploit the potential synergistic activity of some
agents. We observed that the combination of cefadroxil and
amoxicillin (both are b-lactam drugs) was most synergistic
against S. aureus inhibiting more than 80% isolates. Previous
studies also illustrated synergism between two b-lactam antibiotics against different organisms (Gutmann et al., 1986; Pasticci et al., 2008; Jones and Johnson, 1998; Matsumoto,
1998; Tascini et al., 2004; Desbiolles et al., 2001).
Against P. aeruginosa, the combination of cefadroxil and
streptomycin (b-lactam and aminoglycoside) was found to be
most effective, showed more than 80% inhibition. It was also
observed that this combination was also synergistic against
more than 75% isolates of S. aureus.
Gram-negative bacteria such as P. aeruginosa are often
simultaneously isolated from patients with MRSA infections
(Konno, 1995). Therefore, monotherapy with agents with
activities against gram-positive bacteria, such as vancomycin,
is not effective against polymicrobial infections caused by both
MRSA and P. aeruginosa (Shimizu et al., 1996). A better approach to the development of a new antibiotic combination
would be to aim for broad and potent activities against a wide
range of Gram-positive and Gram-negative bacteria including
S. aureus and P. aeruginosa. Our results demonstrated that the
combination of cefadroxil and streptomycin was synergistic
against both S. aureus and P. aeruginosa.
5. Conclusion
Our data clearly state that the use of synergistic combinations
for therapy of polymicrobial infections and infection of multidrug resistant strains is coupled with a better clinical response
than the use of nonsynergistic combinations. Each situation in
which it seems prudent or necessary to use antibiotics in combination must be carefully evaluated. The dangers of toxicity,
sensitization, superinfection, or increase in resistant organisms
that may follow the use of an additional antibiotic, should be
minimized. However, the mortality in severe nosocomial infections is extremely high, especially in debilitated patients, and the
use of synergistic combinations of antibiotics might prove to be
a valuable means for reducing this high mortality rate.
Therefore, when one is dealing with a serious infection presumably caused by S. aureus and P. aeruginosa, it might be useful to
test routinely combinations of antibiotics for a potential synergistic action. Routine use of combinations of antibiotics should,
however, be practiced with caution after prior in vitro trials.
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