(MDRO), clinicians are facing an acute shortage
of antibiotics with activity against the MDROs.
Pathogens like methicillin resistant Staphylococcus
aureus (MRSA), vancomycin resistant enterococci
(VRE) and extended spectrum β lactamase (ESBL)
producing Gram-negative bacilli harbour genetic
determinants, which render them resistant to most
of the available antimicrobials. With the emergence
and spread of carbapenem resistant and metallo- β
lactamase (MBL) producing Pseudomonas aeruginosa
In the present era of multi-drug resistant organisms
Tigecycline susceptibility report from an Indian tertiary care hospital
Bijayini Behera, Anupam Das*, Purva Mathur, Arti Kapil*, Ravisekhar Gadepalli* & Benu Dhawan*
Department of Laboratory Medicine, Jai Prakash Narayan Apex Trauma Centre & *Department of Microbiology
All India Institute of Medical Sciences, New Delhi, India
Received January 31, 2008
Background & objectives: Treatment of serious life threatening infections due to multi-drug resistant
pathogens presents a difficult challenge due to the limited therapeutic options. Therefore, we studied
the in vitro susceptibility of tigecycline, a new glycylcycline with promising broad spectrum of activity
against Gram positive and Gram negative bacteria at a tertiary care hospital in north India.
Methods: A total of 75 multi-drug resistant isolates of methicillin resistant Staphylococcus aureus (21),
vancomycin resistant enterococci (14), vancomycin resistant Streptococcus spp. (3), extended spectrum β
lactamase producing Gram negative bacteria (11) and multi-resistant Acinetobacter spp. (26) were tested
for tigecycline susceptibility by the E- test and disc diffusion methods. An additional 83 multi-resistant
Gram negative clinical isolates were screened by disc diffusion method alone.
Results: All the isolates of MRSA, VRE, vancomycin resistant Streptococcus spp. and ESBL producing
enteric bacteria were sensitive to tigecycline by the E-test and disc diffusion methods. However, only 42
per cent of Acinetobacter spp. were found to be sensitive to tigecycline by the E-test method.
Interpretation & conclusions: In conclusion, tigecycline was found to be highly effective against Gram-
positive bacteria and Gram-negative members of Enterobacteriaceae, but a high prevalence of resistance
in members of Acinetobacter spp. is worrisome.
Key words Acinetobacter spp. - India - multi-drug resistance - tigecycline
and Acinetobacter spp., the only viable treatment
option remains the potentially toxic colistin/polymyxin
B group of antibiotics1. Infections by these MDRO
lead to prolonged hospitalization, increased mortality,
morbidity and cost of treatment.
of antimicrobial agents, glycylcyclines, represented
by tigecycline, a 9-t-butylglyclamide derivative
of minocycline is a significant advancement2-5.
Tigecycline has a spectrum of activity unparalleled by
any other broad spectrum agent and includes MRSA,
In this scenario, the development of a new class
Indian J Med Res 129, April 2009, pp 446-450
VRE, penicillin resistant S. pneumoniae, Haemophilus
gonorrhoeae, ESBL producing
bacteria, MDR Acinetobacter spp., anaerobes and
rapid growing Mycobacterial species2,4,6,. Moreover,
tigecycline is not affected by most of the known
mechanisms of resistance to tetracycline encountered
in bacteria. Because of the promising microbiological,
against clinically important bacteria, tigecycline is
being evaluated for empirical therapy of seriously ill
teaching hospital, the prevalence of antimicrobial
resistance is extremely high across both Gram-
positive and Gram-negative bacterial genera due to the
immense antibiotic pressure8-10. In critical care units,
carbapenems are being used as last resort treatment of
MDR P. aeruginosa and Acinetobacter spp8. Currently,
MDR Acinetobacter spp. remains the most problematic
pathogen in our hospital, especially in the ICUs. In
view of the increasing resistance in both Gram-positive
and Gram-negative pathogens in India and across the
world, this study was conducted to evaluate the in
vitro activity of tigecycline against a contemporary
collection of multiple drug resistant clinical isolates.
At our institute, a 2000 bedded, tertiary care and
Material & Methods
Bacterial isolates: The study was conducted in two
bacterial strains kept in the Bacteriology laboratory of
All India Institute of Medical sciences (AIIMS), New
Delhi were used. In this part, tigecycline susceptibility
testing was done on confirmed isolates of VRE,
vancomycin resistant Streptococcus spp., MRSA,
ESBL-positive Gram-negative bacteria (GNB) and
MDR Acinetobacter spp. Identification of these isolates
was done using routine microbiological methods and
VITEK 2 (BioMérieux, Hazelwood, France) system.
In general, antimicrobial susceptibility testing of all
these isolates was done by the disc diffusion method
according to the Clinical and Laboratory Standards
Institute (CLSI) guidelines11 and the VITEK-2 system.
Specifically, for S. aureus, screening for methicillin
resistance was done by the oxacillin agar screen
method using 6 µg/ml oxacillin (Sigma, USA) as
recommended by the CLSI11. All the isolates were also
screened for methicillin resistance by the cefoxitin
(30 µg) disc method12. The S. aureus ATCC 25923 (Hi
For the first part of the study, stocks of confirmed
Media, Mumbai, India) and WHO-213 were used as
negative and positive controls respectively for MRSA
screening. Confirmation of methicillin resistance was
done by demonstrating the presence of mecA gene
by PCR using published primers14. For enterococci,
screening for vancomycin resistance was done by the
agar screen methods on Mueller-Hinton agar (MHA)
(BBLTM BD, USA) using 6 µg/ml vancomycin (Hi-
Media Laboratories, Mumbai, India)15. Vancomycin
resistance was confirmed by determining the minimum
inhibitory concentration (MIC) of vancomycin by
E test (AB Biodisk, Sweden) and Vitek 2 system15.
Enterococcus faecalis ATCC 29212 (Hi-Media,
Mumbai, India) was used as control. All Gram
negative isolates were screened for ESBL production
by the disk potentiation test using ceftazidime (CAZ)
and ceftazidime + clavulinic acid (CAZ+clav) disc
(BBL TM BD, USA)11. ESBL E- test (CAZ/CAZ+clav)
(AB Biodisk, Sweden) was also performed according
to manufacturer’s instructions to confirm the presence
of clavulanic acid inhibitable ESBLs16. Escherichia
coli ATCC 25922 and Klebsiella pneumoniae ATCC
700603 were used as negative and positive controls
respectively for ESBL testing. Further, screening
for MBL production was done in all isolates of
Acinetobacter spp. by the imipenem- EDTA combined
disk test. The test was performed according to published
protocols17. Isolates with a positive EDTA-imipenem
disc synergy test were subsequently evaluated by the
MBL E-test strip containing a double sided seven-
dilution range of imipenem (IP) (4 to 256 µg/ml) and
imipenem (1 to 64 µg/ml) in combination with a fixed
concentration of EDTA (320 µg/ml) (IPI)18. MIC ratio
of IP/IPI of > 8 or > 3 log2 dilutions was taken as MBL
Tigecycline susceptibility testing: In the first part
of the study, tigecycline MIC was determined for
all MRSA, VRE, ESBL producing GNB and MDR
Acinetobacter spp. using the E-test (AB Biodisk,
Sweden) method according to the manufacturer’s
instructions. For tigecycline, the MIC breakpoints
used for susceptibility were taken as ≤ 0.5 µg/ml for
S. aureus, ≤ 0.25 µg/ml for enterococci, and ≤ 2 µg/
ml for Gram-negative bacteria, as approved by the
FDA2,5,19. Disc diffusion susceptibility testing was
also performed for all these isolates using tigecycline
disks (15 µg; Oxoid Ltd, Basingstoke, Hants, UK). The
interpretation of zone diameters for all Gram negative
bacteria (including Acinetobacter spp.) was done using
the US FDA tigecycline susceptible breakpoints listed
BEHERA et al: TIGECYCLINE SUSCEPTIBILITY IN INDIA 447
for Enterobacteriaceae (MIC ≤ 2 µg/ml, and ≥ 19
mm zone size)5. Resistance was defined as MIC ≥ 8
µg/ml and zone size ≤ 14 mm18. Interpretation of zone
diameters of all Gram-positive bacteria was done using
the US FDA tigecycline susceptible breakpoints listed
for S. aureus (MIC ≤ 0.5 µg/ml and ≥ 19 mm zone size)
and E. faecalis (vancomycin susceptible only) (MIC ≤
0.25 µg/ml and ≥ 19 mm zone size)20.
clinical isolates of Gram-negative bacteria (E coli,
Klebsiella spp. and Acinetobacter spp.) obtained over
a period of two months (July-August 2007) at the
Microbiology laboratory of Trauma Centre, AIIMS,
New Delhi, which were resistant to two or more of
the most commonly used antimicrobial classes for
the treatment of the indicated infection (MDR) were
included. Antibiotic sensitivity patterns of these
isolates were compared in order to exclude clonal
origin of the clinical isolates. Only one isolate per
patient was included for the study. Screening for
tigecycline susceptibility in these isolates was done
by the disc diffusion method alone, using tigecycline
disks (15 µg; Oxoid Ltd., Basingstoke, Hants, UK).
For the second part of the study, consecutive
Results & Discussion
in the first part of the study. These included 21 confirmed
isolates of MRSA, 14 of VRE (vancomycin MIC >256
µg/ml), three of vancomycin resistant Streptococcus
uberis (vancomycin MIC> 256 µg/ml), 11 isolates
of ESBL producing members of enterobacteriaceae
(6 K. pneumoniae and 5 E. coli) and 26 of MDR
Acinetobacter spp. Since tigecycline has no or limited
activity against Pseudomonas and Proteus spp., these
were not included in our study.
A total of 75 MDR clinical isolates were evaluated
by PCR, showing a 310 base pair size band, using
published primers14. All of them were found to be
sensitive to tigecycline by the E-test method. The
MIC50 and MIC90 of the isolates of MRSA in our study
was found to be 0.25 and 0.38 µg/ml respectively,
much below the US FDA cut-offs for susceptibility.
The isolates had a zone diameter of tigecycline by disc
diffusion method varying from 23-30 mm. Of the 14
isolates of VRE, 13 were E. faecium and one was E.
gallinarum. The MIC50 and MIC90 of these isolates
was found to be respectively 0.064 and 0.094 µg/
ml. All these isolates had tigecycline zone diameters
ranging from 24-38 mm by the disk diffusion method.
The relatively low MICs of tigecycline for Gram-
All the isolates of MRSA were mecA positive
positive cocci reflect their unexposed status to this new
found to be sensitive by the E-test and disc diffusion
method. The MIC50 and MIC90 of these isolates were
found to be 0.38 and 0.75 µg/ml respectively. The zone
diameter of tigecycline in these isolates varied from
22-29 mm. Thus, tigecycline was sensitive against all
isolates of MDR Gram positive and Gram negative
enteric bacteria by the E-test and disk diffusion
method in the first part of this study. Isolates of ESBL
producing members of Enterobacteriaceae having a
low MIC for tigecycline signifies its potential utility
in clinical infections due to these notorious MDR
All the 11 MDR K. pneumoniae and E coli were
included in the first part, all of which were MDR.
Additionally, carbapenem resistance was seen in 20
(77%) isolates, of which 17 (85%) were found to
be MBL producers. MBL production was confirmed
by the imipenem-EDTA combined disc test and E-
test methods, both of which showed 100 per cent
concordance. Of the 26 isolates of A. baumannii,
11 (42%) had a tigecycline MIC of ≤ 2 µg/ml by
the E-test and were deemed susceptible according
to US FDA breakpoints; two (7.6 %) had an MIC of
8 µg/ml (resistant) and 13 (50%) had MICs ranging
between 3-6 µg/ml (intermediate). All the isolates
having MIC ≤ 2 µg/ml also had a zone diameter ≥
19 mm (cut-off for susceptibility). However, of the
two isolates showing MIC of 8 µg/ml, only one was
found to be resistant by the disk diffusion method
(zone diameter 14 mm). Similarly, of the 13 isolates
found to have MICs ranging from 3-6 µg/ml, 7
(54%) were intermediate (15-18 mm), 5 (38%) were
sensitive (≥ 19 mm) and one was resistant (≤ 14 mm)
by the disc diffusion method. When a cut-off of ≥ 13
mm was considered as breakpoint for susceptibility
as recommended by some authors22, all the isolates
were found to be sensitive by disk diffusion method.
The tigecycline zone diameter for A. baumannii by
disc diffusion method ranged from 14-26 mm. Thus,
we found discordance in the results of E-test and
disc diffusion susceptibility testing methods in A.
baumannii. With the interpretative zone diameters of
> 19 mm for sensitive, 17 (65 %) isolates were
sensitive, 7 (27%) were intermediate and 2 (7.6 %)
were resistant; with the interpretative zone diameter
of ≥ 13 mm22, all the isolates of A. baumannii were
sensitive by the disc diffusion method. We also found
A total of 26 A. baumannii isolates were
448 INDIAN J MED RES, APRIL 2009
a gradual reduction of tigecycline zone diameter as the
MICs for the Acinetobacter isolates increased (Fig.).
MDR Gram-negative clinical isolates were screened
for tigecycline susceptibility by the disc diffusion
method over a period of two months. Only one isolate
per patient was included. The antibiogram of the
isolates differed, signifying their non-clonal origin.
These included a total of 25 isolates of ESBL producing
Enterobacteriaceae (16 E. coli and 9 Klebsiella spp.)
and 58 isolates of MDR Acinetobacter spp. All the
isolates of E. coli and Klebsiella spp. were found to be
sensitive to tigecycline by the disk diffusion method.
However, of the 58 isolates of Acinetobacter spp., only
22 (38%) had a zone diameter ≥ 19 mm; 21 (36%) had
a diameter between 15-18 mm and 15 (26%) had a zone
diameter of ≤ 14 mm. When a zone diameter of ≥ 13
mm was considered as breakpoint for susceptibility22,
13 (22%) isolates were found to be resistant by the disc
diffusion screening. We could not perform E-test on
these isolates, therefore no association could be drawn
about the MICs.
In the second part of the study, 83 consecutive
amongst Acinetobacter spp. in our study is worrisome
since the organism is not only totally unexposed
to tigecycline but also to the tetracycline group of
antibiotics in our hospital. Although tigecycline is
A high prevalence of tigecycline resistance
generally effective against MDR Acinetobacter
spp.2,4, a recent report has found an unusually high
rate of tigecycline resistance in these organisms23.
These authors had also used the E-test method, which
correlated 100 per cent with inhibition zone diameters.
In our study, E test reported a higher rate of resistant
and intermediate results as compared to disc diffusion
method. This is again in contrast to another study,
which found an unacceptably high minor error rate
(false intermediate results) by the disc diffusion for
Acinetobacter isolates with tigecycline MIC of 1 or
2 µg/ml19. The tigecycline evaluation test (TEST), a
global programme, is reported to have only 2 per cent
of the 4247 Acinetobacter isolates showing tigecycline
MIC ≥ 2 µg/ml24. However, very limited clinical
data are available to draw conclusion on the utility
of tigecycline in treatment of MDR Acinetobacter
inter-method agreement of tigecycline in vitro
susceptibility testing so that breakpoints and
disc diffusion guidelines can be formulated for
Acinetobacter spp. This will minimize interpretative
difference amongst various studies and provide the
true magnitude of resistance in this genus. Reporting
of tigecycline resistance in Acinetobacter from two
geographically distinct locations may have important
therapeutic implications where it has become a last
resort antimicrobial. Since tigecycline has a long
terminal half-life and a large volume of distribution,
it can be used as a life saving antimicrobial in
polymicrobial infections due to Gram-positive and
enteric Gram-negative bacteria7. However, its use
needs to be strictly monitored to prevent development
and dissemination of resistance against this one of the
last available antimicrobial molecules.
More studies are needed to investigate the
the tigecycline discs used for disc diffusion testing in this study.
Authors acknowledge Wyeth Pharmaceuticals for providing
Conflicts of interest - None
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Reprint requests: Dr Purva Mathur, Assistant Professor, Department of Laboratory Medicine, Jai Prakash Narayan Apex Trauma Centre
All India Institute of Medical Sciences, New Delhi 110 029, India
450 INDIAN J MED RES, APRIL 2009