Multi-drug resistance in Acinetobacter baumannii strains isolated from clinical specimens from three hospitals in Tehran-Iran

Abstract

The aim of this study is to investigate multi-drug resistance in A. baumannii strains that was isolated from clinical samples of three highly large hospitals in Tehran-Iran. This descriptive-cross sectional study was performed in three large hospitals in Tehran on 70 samples of Acinetobacter which were isolated from patients during April to November 2010. After identifying the species level by using culture and biochemical methods, in order to determine sensitivity of 50 isolates of A. baumannii to 13 antibiotics, standard methods according to CLSI guidelines were performed. In this study, resistance to three or more of three classes of antibiotics multidrug resistance was defined. In this study, 50 A. baumannii strains, 12 A. lwoffii strains and other Acinetobacter species were isolated from patients. The majority of isolates were from blood specimens. Isolates of A. baumannii showed the highest resistance to cefepime, ceftazidime, aztreonam, norfloxacin, ofloxacin, ciprofloxacin and amikacin. Tobramycin and meropenem considered as effective drugs in this study. Multi-drug resistance in these strains was respectively 55.4%. Multi-drug resistant Acinetobacters are growing and considered as important threat for hospitalized patients, so change in consumption patterns of antibiotics and control of hospital infections seems to be necessary.

Full text

African Journal of Microbiology Research Vol. 5(21), pp. 3579-3982, 9 October, 2011
Available online http://www.academicjournals.org/ajmr
DOI: 10.5897/AJMR11.159
ISSN 1996-0808 ©2011 Academic Journals
Full Length Research Paper
Multi-drug resistance in Acinetobacter baumannii
strains isolated from clinical specimens from three
hospitals in Tehran-Iran
Sepideh Mostofi1, Reza Mirnejad2* and Faramaz Masjedian3
1Department of Biology, North Tehran Azad University, Tehran, Iran.
2Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
3Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Accepted 23 June, 2011
The aim of this study is to investigate multi-drug resistance in A. baumannii strains that was isolated
from clinical samples of three highly large hospitals in Tehran-Iran. This descriptive-cross sectional
study was performed in three large hospitals in Tehran on 70 samples of Acinetobacter which were
isolated from patients during April to November 2010. After identifying the species level by using
culture and biochemical methods, in order to determine sensitivity of 50 isolates of A. baumannii to 13
antibiotics, standard methods according to CLSI guidelines were performed. In this study, resistance to
three or more of three classes of antibiotics multidrug resistance was defined. In this study, 50 A.
baumannii strains, 12 A. lwoffii strains and other Acinetobacter species were isolated from patients.
The majority of isolates were from blood specimens. Isolates of A. baumannii showed the highest
resistance to cefepime, ceftazidime, aztreonam, norfloxacin, ofloxacin, ciprofloxacin and amikacin.
Tobramycin and meropenem considered as effective drugs in this study. Multi-drug resistance in these
strains was respectively 55.4%. Multi-drug resistant Acinetobacters are growing and considered as
important threat for hospitalized patients, so change in consumption patterns of antibiotics and control
of hospital infections seems to be necessary.
Key words: Acinetobacter baumannii, nosocomial infection, multi-drug resistance, antibiogram.
INTRODUCTION
The genus Acinetobacter comprises Gram negative,
oxidase negative, strictly aerobic and non motile bacteria.
Various species of Acinetobacter are widespread in
nature. They can be recovered from virtually all samples
obtained from soil, surface water, human skin, food and
waste (Gaynes et al., 2005; Leung et al., 2006; Peleg et
al., 2008).
The genus known as Acinetobacter has significant
taxonomic modification over last 30 years. Acinetobacter
baumannii is the most common species that isolates from
patients and other species such as A. lwoffii, A.
Johnsonii, and A. haemolyticus are rarely isolated from
*Corresponding author. E-mail: rmirnejadreza @ yahoo.com.
Tel/Fax: +98(21)88039883.
patients (Peleg et al., 2008).
Nowadays, due to A. baumannii significant clinical
properties and its ability to achieve drug resistance, it
considered as one of the microorganisms that threaten
antimicrobial medication. Acinetobacter baumannii
causes nosocomial infections such as bacteremia,
urinary tract infections and secondary meningitis, but it
has prominent role in creation of hospital pneumonia
especially pneumonia that acquired in upper respiratory
tract in patients that hospitalized in intensive care units
(ICU) (Anstey et al., 2002; Dijkshoorn et al., 2005;
Fournier and Richet, 2006; Peleg et al., 2008; Rizos et
al., 2007; Villers et al., 1998).
Different studies demonstrated that various species of
A. baumannii are resistant to wide range of antibiotics.
Spread of multi-drug resistant A. baumannii is not limited
to hospitals of one city, but is also important in national

3580 Afr. J. Microbiol. Res.
scale (Metan et al., 2007; Unal et al., 2005; Wisplinghoff
et al., 2007; Wroblewska et al., 2007). Since several
factors causing resistance in A. baumannii, treatment of
infections caused by this organism should be based on
perfect antibiotics sensitivity tests, therefore having
information regarding the prevalence and pattern of
bacterial resistance to these drugs is important (Halstead
et al., 2007; Scott et al., 2007; Van Dessel et al., 2004).
In this study is investigation of multi-drug resistance in A.
baumannii strains that isolated from clinical samples from
three large hospitals in Tehran-Iran.
MATERIALS AND METHODS
This descriptive-cross sectional study was conducted from April to
November 2010. Five hundred samples of blood, respiratory
sections, urine, skin sores, and trachea were collected from patients
of three large hospitals of Imam Khomeini, Milad, Baqiyatallah and
transferred to the laboratory by BHI broth medium.
In the laboratory each sample was cultured on blood agar and
MacConkey agar (Merck Co., Germany) and incubated for 24 h in
37°C. Blood specimens was cultured in Trypticase Soy Broth (TSB)
(Merck, Germany) and sub-cultured on chocolate agar. After 24 h,
with direct examination (Gram staining) presence of gram negative
coccobacillus was confirmed by the microscopic approach. In order
to recognize different species of Acinetobacter, all suspected
colonies were identified by colonial morphology, Gram-staining,
positive catalase, negative oxidase, growth in 37 and 42°C and
other biochemical reactions.
After identification of Acinetobacter species, in order to determine
the drug resistance phenotype, disk diffusion method as recom-
mended by clinical laboratory and standards institute (CLSI) was
performed. In this study 13 different antibiotic disks from Oxoid Ltd.
(Basingstoke, UK) were used which included amikacin (30 μg),
ampicillin/sulbactam (10/10 μg), aztreonam (30 μg), cefepime (30
μg), ceftazidime (30 μg), ciprofloxacin (5 μ), gentamycin(10 μg),
imipenem (10 μg), meropenem (10 μg), norfloxacin (10 μg),
ofloxacin (1 μg), piperacillin / tazobactam (100/10 μg), tobramycin
(10 μg). In addition, the antibiotic potency of the disks was
standardized against the reference strains of E. coli (ATCC 25922)
as negative control and the reference strain of A. baumannii (ATCC
19606) as positive control were used.
It is to say according to studies, isolates of A. baumannii that
show resistance to three or more than three categories, including
quinolone antibiotics (ciprofloxacin), broad spectrum
cephalosporins (ceftazidime and cefepime), combined
lactam/lactamase inhibitor(Ampicillin/sulbactam), aminoglycosides
(amikacin, tobramycin) and carbapenems( imipenem, meropenem)
considered as multi-drug resistant strains. Finally, for statistical
analysis, data were entered into a database using SPSS 16 for
Windows (SPSS Inc., Chicago, IL) and then, results by using Chi-
square test were analyzed.
RESULTS
During this study, a total of 70 samples of Acinetobacter
were isolated from 500 collected samples. Fifty samples
of patients were identified as A. baumannii (71%), 12
samples were A. lwoffii (17.1%) and 8 samples (11.4%)
were other Acinetobacter species.
Results showed that 50 samples of A. baumannii were
isolated from 19 blood samples (38%), 15 trachea tube
aspiration samples (30%), 6 wound samples (12%), 4
urine samples (8%), 1 oral sample (2%), and 5 samples
(10%) had unknown origin.
The majority of strains were isolated from ICUs (20/50)
and the remaining strains were from infectious ward
(15/50), emergency ward (10/50) and other wards.
In this study approximately all samples were resistant
to ceftazidime and cefepime and high resistance to
aztreonam, norfloxacin, ciprofloxacin, amikacin,
imipenem, gentamycin, and ampicillin-sulbactam were
observed. Tobramycin and meropenem considered as
effective drugs in this review. (In Table 1 patterns of
antibiotic resistance in A. baumannii are showed).
Results of this study showed that 27 samples of A.
baumannii (54%) are resistant to three or more than three
antibiotics (Table 2) and 16 samples (32%) showed
resistance to two antibiotics. Also none of resistant
strains were showed complete resistance to all
antibiotics.
Conclusion
Acinetobacter baumannii is an important opportunistic
pathogen to high virulence. It is responsible for severe
nosocomial infections over the last 30 years. This
bacterium, particularly multi-drug resistant strains has
been implicated as the cause of serious infectious
disease in different parts of the hospitals and treatment of
such infections because of their broad resistance to
antibiotics is difficult (Anstey et al., 2002; Gaynes et al.,
2005; Leung et al., 2006; Peleg et al., 2008). Moreover,
since environmental factors and different patterns of
antimicrobial agents play important role in creation and
expansion of these strains in different parts of the world,
in this study, the incidence of multi-drug resistant A.
baumannii from clinical samples of three hospitals in
Tehran-Iran was performed.
In this investigation 71.5% of isolates were A.
baumannii and 28.5% were identified as A. lwoffii and
other Acinetobacter species. A similar finding was also
observed by Constantiniu and colleagues during years
2001 to 2004. They from 24 clinical isolates, 71% A.
baumannii and 29% A. lwoffii were detected
(Constantiniu et al., 2004).
Hujer et al. (2006) in their study that was conducted on
military and civilian patients in Iraq and Afghanistan
reported that 15% of the strains were resistant to all nine
antibiotics which were tested and 89% of strains showed
resistance to at least three antibiotic classes. In their
study more than 90% of isolates were resistant to
ciprofloxacin, less than 80% to cephalosporins with a
broad spectrum, 40% to ampicillin-sulbactam, 20% to
imipenem and 81% to at least one of the
aminoglycosides (amikacin or tobramycin) (Hujer et al.,
2006). In this study, more than 90% of isolates to

Mostofi et al. 3581
Table 1. Frequency of antibiotic resistance in A. baumannii isolates.
Antibiotic
Sensitivity patterns (%)
Resistant
Intermediate
Sensitive
Cefepime
96
4
0
Ceftazidime
96
4
0
Aztreonam
95
3
2
Norfloxacin
95
1
4
Ofloxacin
88
4
8
Ciprofloxacin
88
4
8
Amikacin
85
5
10
Imipenem
76
2
22
Gentamycin
61
3
36
Ampicillin-sulbactam
59
3
38
Piperacillin-tazobactam
40
8
52
Meropenem
31
4
65
Tobramycin
26
2
72
Table 2. Frequency of multi- drug resistance in A. baumannii.
Parameter
Resistance to one or several antibiotics
Total
Number of antibiotics
1
2
3
4
>4
50
Number of isolates resistant A. baumannii
7
16
13
5
9
ciprofloxacin, ofloxacin, and cephalosporins (ceftazidime
and cefepime), 76% to imipenem, 59% to ampicillin /
sulbactam, and less than 35% to meropenem and
tobramycin were resistant (Table 1). Also in this study
more than 50% of isolates were resistant to at least three
antibiotic classes (Table 2). Differences observed
between two studies could be due to methods and
resistance patterns that are influenced by environmental
factors and antimicrobial patterns which were used.
Necessary to say, international travels are also important
in development of multi-drug resistant strains.
Acinetobacter strains with antibiotic resistance have
been reported from all around the world (Brink et al.,
2007). In the study in year 2003 that performed by Ayan
and colleagues, of 52 strains, all isolates were resistant
to piperacillin, piperacillin-tazobactam, ticarcillin-
clavulanic acid, cefepime, cefotaxime, ceftazidime,
ceftriaxone, gentamycin and resistant to tobramycin ,
ciprofloxacin, ampicillin-sulbactam, co-trimoxazole, and
amikacin that results highly conform with results of this
research (Ayan et al., 2003).
Rahbar and colleagues in the year 2005 to 2006 were
determined that, A. baumannii shows high percentage of
resistance to ceftriaxone (90.9%), piperacillin (90.9%),
ceftazidime (84.1%), amikacin (85.2%),and ciprofloxacin
(90.9%) that results partly conform with results of this
research. They had also conducted that imipenem was
the most effective agent against these organisms
(resistance 4.5%) that is in conflict with our results
(Rahbar et al., 2010).
In a study that in year 1998 to 2001 was conducted by
Karlowsky et al., 90% of A. baumannii strains were
sensitive to meropenem, but in this investigation, only
44% of strains showed resistance to meropenem and
usage of this antibiotic with tobramycin could be more
effective against A. baumannii strains (Karlowsky et al.,
2003).
In a study that performed by Hoe Koo et al in years
2007 to 2008, they determined amikacin as the most
effective drug among nine antimicrobial agents, unlike, in
this study tobramycin and imipenem were the most
effective agents among 11 antimicrobial agents which
were used (Hoe Koo et al., 2010).
Overall results indicate that among common
Acinetobacter species, A. baumannii is more responsible
for nosocomial infections and more than 50% of strains
are multi-drug resistant, so control of hospital infections
seems to be necessary among the three hospitals which
were investigated. In this regard, in response to
uncontrolled use of antibiotics, multi-drug resistant A.
baumannii in hospital environment increased, so control
of antibiotics usage in hospitals play an important role in
preventing the emergence of such strains and infections
caused by them.

3582 Afr. J. Microbiol. Res.
ACKNOWLEDGMENTS
This study was supported by cell and molecular biology
research center, the authors also appreciate the
microbiology group of Tehran Medicine University for
their sponsorship and financial supports.
REFERENCES
Anstey NM, Currie BJ, Hassell M, Palmer D, Dwyer B, Seifert H (2002).
Community-acquired bacteremia Acinetobacter pneumonia in tropical
Australia is caused by diverse strains of Acinetobacter baumannii,
with carriage in the throat in at-risk groups. J. Clin. Microbiol.,
40:685–686.
Ayan M, Durmaz R, Aktas E, Durmaz B (2003). Bacteriological, clinical
and epidemiological characteristics of hospital-acquired
Acinetobacter baumannii infection in a teaching hospital. J. Hosp.
Infect., 54:39-45.
Brink A, Moolman J, Da Silva MC, Botha M (2007). Antimicrobial
susceptibility profile of selected bacteraemic pathogens from private
institutions in South Africa. S. Afr. Med. J., 97:273–279.
Constantiniu Sofia, Romaniuc, A, Iancu SL, Filamon R, Tarasi I (2004).
Cultural and biochemical characteristics of Acinetobacter spp. Strains
isolated from hospital units. J. Preven. Med., 12:35-42.
Dijkshoorn L, Van Aken E, Shunburne L, Van Der Reijden TJ, Bernards
AT, Nemec A, Towner KJ (2005). Prevalence of Acinetobacter
baumannii and other Acinetobacter spp. In faecal samples from non-
hospitalised individuals. Clin. Microbiol. Infect., 11:329–332.
Fournier PE, Richet H (2006). The epidemiology and control of
Acinetobacter baumannii in health care facilities. Clin. Infect. Dis., 42:
692–699.
Gaynes R, Edwards JR (2005). Overview of nosocomial infections
caused by Gram-negative bacilli. Clin. Infect. Dis., 41:848–854.
Halstead DC, Abid J, Dowzicky MJ (2007). Antimicrobial susceptibility
among Acinetobacter calcoaceticus-baumannii complex and
Enterobacteriaceae collected as part of the tigecycline evaluation and
surveillance trial. J. Infect., 55: 49–57.
Hoe Koo S, Kwon KC, Cho HH, Sung JY (2010). Genetic Basis of
multidrug-resistant Acinetobacter baumannii clinical isolates from
three university hospitals in Chungcheong Province, Korea. Korean J.
Lab. Med., 30:498-506.
Hujer KM, Hujer AM, Hulten EA, Bajaksouzian S, Adams JM, Donskey
CJ, Ecker DJ, Massire C, Eshoo MW, Sampath R, Thomson JM,
Rather PN, Craft DW, Fishbain JT, Ewell AJ, Jacobs MR, Paterson
DL, Bonomo RA (2006). Analysis of antibiotic resistance genes in
multidrug-resistant Acinetobacter spp. Isolates from military and
civilian patients treated at the Walter Reed Army Medical Center.
Antimicrob. Agents Chemother., 50:4114–4123.
Karlowsky J, Draghi D, Jones M, Thornsberry C, Friedland I, Sahm D
(2003). Surveillance for antimicrobial susceptibility among clinical
isolates of Pseudomonas aeruginosa and Acinetobacter baumannii
from hospitalized patients in the United States, 1998 to 2001.
Antimicrobial. Agents Chemother., 47:1681-1688.
Leung WS, Chu CM, Tsang KY, Lo FH, Lo KF, Ho PL (2006). Fulminate
community-acquired Acinetobacter baumannii pneumonia as a
distinct clinical syndrome. Chest., 129:102–109.
Metan G, Alp E, Aygen B, Sumerkan B (2007). Carbapenem-resistant
Acinetobacter baumannii, an emerging threat for patients with post-
neurosurgical meningitis. Int. J. Antimicrob. Agents, 29: 112–113.
Peleg AP, Seifert H, Paterson DL (2008). Acinetobacter baumannii:
Emergence of a Successful Pathogen. Clin. Microbiol. Rev., 21:538-
582.
Rahbar M, Mehrgan H, Aliakbari NH (2010). Prevalence of antibiotic-
resistant Acinetobacter baumannii in a 1000-bed tertiary care hospital
in Tehran, Iran. Indian J. Pathol. Microbiol., 53:290-293.
Rizos I, Tsiodras S, Papathanasiou S, Rigopoulos A, Barbetseas J,
Stefanadis C (2007). Prosthetic valve endocarditis due to
Acinetobacter Spp, a rare case and literature review. Am. J. Med.
Sci., 333:197–199.
Scott P, Deye G, Srinivasan A, Murray C, Moran K, Hulten E, Fishbain
J, Craft D, Riddell S, Lindler L, Mancuso J, Milstrey E, Bautista CT,
Patel J, Ewell A, Hamilton T, Gaddy C, Tenney M, Christopher G,
Petersen K, Endy T, Petruccelli B (2007). An outbreak of multidrug-
resistant Acinetobacter baumannii-calcoaceticus complex infection in
the US military health care system associated with military operations
in Iraq. Clin. Infect. Dis., 44: 1577–1584.
Unal S, Garcia-Rodriguez JA (2005). Activity of meropenem and
comparators against Pseudomonas aeruginosa and Acinetobacter
spp. Isolated in the MYSTIC Program, 2002–2004. Diagn. Microbiol.
Infect. Dis., 53:265–271.
Van Dessel H, Dijkshoorn L, van der Reijden T, Bakker N, Paauw A,
van den Broek P, Verhoef J, Brisse S (2004). Identification of a new
geographically widespread multiresistant Acinetobacter baumannii
clone from European hospitals. Res. Microbiol., 155:105–112.
Villers D, Espaze E, Coste-Burel M, Giauffret F, Ninin E, Nicolas F,
Richet H (1998).Nosocomial Acinetobacter baumannii infections,
microbiological and clinical epidemiology. Ann. Intern. Med., 129:
182–189.
Wisplinghoff H, Schmitt R, Wohrmann A, Stefanik D, Seifert H (2007).
Resistance to disinfectants in epidemiologically defined clinical
isolates of Acinetobacter baumannii. J. Hosp. Infect., 66:174–181.
Wroblewska MM, Towner KJ, Marchel H, Luczak M (2007). Emergence
and spread of carbapenem-resistant strains of Acinetobacter
baumannii in a tertiary-care hospital in Poland. Clin. Microbiol. Infect.,
13:490–496.