Klebsiella sp. is a group of short, non-motile, gram negative
rods and they are present in the respiratory tract and feces of about
5% of normal individuals. They can cause different kinds of infections,
for example, pneumonia, urinary tract infection and bacteremia in immunocompromised
hosts. K. pneumoniae and K. oxytoca cause hospital- acquired
infections (Brooks et al., 2004).
Multi-drug resistant Klebsiella has been recognized as a cause
of hospital acquired infections worldwide (Eisen et al., 1995;
Keynan and Rubinstein, 2007). They are resistant to numerous antibiotics,
including aminoglycosides, penicillins and extended spectrum cephalosporins.
Their resistance to antibiotics restricts the choice of antibiotics for
therapy (Hogg et al., 1993).
There are different mechanisms for development of resistance in Klebsiella
sp. including the acquisition of plasmids which code for the production
of extended spectrum beta-lactamases and aminoglycoside modifying enzymes.
The genetic origin of drug resistance may be plasmid or chromosomal.
Plasmids carry genes for resistance to often more than one antibiotics,
these plasmids so-called R-plasmids. R-plasmids can easily transfer between
different species and even genera of bacteria, so, determination of the
genetic origin of resistance in clinical isolates of bacteria can predict
the rate of transferring resistance between clinical isolates (Oktem et
Investigation of the plasmid profiles of nosocomial isolates and determination
of the pattern of restriction enzyme analysis of plasmids are reliable
methods for typing of hospital bacterial isolates, however, there is no
data in the literature about plasmid profiles and pattern of restriction
enzyme analysis of plasmids of Klebsiella sp. about clinical isolates
in hospitals of Urmia, North-West of Iran.
In this study, we investigated antibiotic resistance pattern, plasmid
profiles and patterns of restriction enzyme analysis of plasmids in clinical
isolates of Klebsiella sp. As well as relation between plasmid
pattern and resistance to different antibiotics were determined.
MATERIALS AND METHODS
Bacterial isolates: A total of 39 isolates were collected from
urine specimens submitted to two educational hospital clinical laboratories
in Urmia/Iran during a three months period from December 2006 until March
2007. The isolates were further processed by the standard methods to identify as
the Klebsiella sp. In brief some of biochemical tests were
used for identification of the isolated bacteria as Klebsiella sp.
was indole production, methyl red, voges-proskauer, citrate, SH2
production, urea hydrolysis, Phenylalanine deaminase, Lysine decarboxylase,
Arginine dihydrolase, motility and several sugars fermentation (Baron
and Finegold, 1990). Isolated bacteria were maintained for long storage
on skimmed milk medium (BBL) by adding 10% glycerol in -60°C, cultures
were maintained for daily use on Nutrient agar (BBL) slants on 4°C.
Susceptibility testing: The Muller Hinton Agar (MHA) and Muller
Hinton Broth (MHB) media (Merck) were used for detection of antibiotic
resistance of isolates. The susceptibility of isolates to different antibiotics
was tested using agar disk diffusion method (Bauer et al., 1966;
Jazani et al., 2007). To represents different classes of antibacterial
agents commonly used for treatment of Klebsiella infections, gentamicin,
tobramycin, ceftizoxime, co-trimoxazole, amikacin, cephtazidime, ciprofloxacin,
kanamycin, nalidixic acid, ampicillin and nitrofurantoin were used in
present study (Hi-media, Mombay, India).
Extraction, purification and electrophoresis of plasmids: Plasmids
were extracted by alkaline lysis method with some modifications. In brief,
isolates were cultured in LB medium and after centrifugation, cellular
pellet were re-suspended in 100 μL of GTE buffer (2 mL EDTA 0.5 M,
10 mL Glucose 0.5 M and 2.5 mL Tris-base 1 M pH = 8). Two hundred microliter
of lysis buffer (1 mL NaOH 1 M and 0.5 mL SDS 10%) were added and tubes
were incubated in ice for 5 min. Then cold sodium acetate solution was
added and tubes were incubated in ice for 10 min, after centrifugation.
Phenol/chloroform/isoamyl alcohol solution in a 1:24:25 ratio was added
to supernatant and after another round of centrifugation, isopropanol
was added to supernatant and samples were centrifuged for 15 min, resulting
white pellet washed with cold ethanol and after centrifugation supernatant
was discarded and micro tubes allowed to dry, pellet solved in 50 μL
of buffer (Tris-HCl pH = 8 10 mM, EDTA 1 mM and RNase) (Sambrook and Russel,
To run electrophoresis 5 μL of loading buffer (Takara, japan) was
added to 35 μL plasmid solution and then 20 μL of aliquots was
loaded on 0.8% agarose gel(contained ethidium bromide). Plasmids were
separated by agarose gel electrophoresis at 45 V for 4-5 h and visualized
under UV illumination.
Restriction digestion of plasmid DNA: Single digestion of plasmid
DNA extracts with EcoRI (Fermentase) and HincII (Cinnagen,
Tehran, Iran) were performed as recommended by the manufacture. The cleavage
products of single digestion were electrophoresed on 1% agarose gel. The
restriction patterns of different plasmid DNA extracts were compared using
DNA ladder (100-12000) as a molecular size marker.
Statistical analysis: Fisher`s exact statistical test was used
for analysis of qualitative data. p-value was considered significant if
RESULTS AND DISCUSSION
Sensitivity of bacterial species to antibiotics: Based on the
results of drug susceptibility testing, 16 different antibiotypes numbered
from 1 to 16 were found. Antibiotype 1 consisted of 8 isolates that showed
resistance to all investigated antibiotics. It was the most prevalent
antibiotype among the isolates (20.5%). Four isolates were sensitive to
all tested antibiotics (antibiotype 2). The remaining antibiotypes consisted
of 27 isolates were resistant to different antibiotics. Ampicillin (79.5%)
and kanamycin (53.8%) showed the highest rate of resistance and ciprofloxacin
and nalidixic acid (30.8%) demonstrated the lowest (Table
The list of 22 MDR phenotypes identified is shown in Table
2, with majority showing resistance to the 11 antimicrobials.
Plasmid profile analysis indicated that ten isolates (25.6%) harbored
one plasmid with identical patterns in all isolates (Fig.
1). The restriction pattern of the ten isolates is identical after
single digestion of plasmids with HincII or EcoRI.
||The rates of resistance to different antibiotics for
39 isolates of Klebsiella from two educational hospitals in
a six months period
|Gentamicin (Gm), Tobramycin (Tob), Ceftizoxime (Ct),
Co-trimoxazole (SXT), Amikacin (AN), Cephtazidime (CAZ), Ciprofloxacin
(Cp), Kanamycin (K), Nalidixic Acid (NA), Ampicillin (Am) and Nitrofurantoin
||Antibiotic resistance phenotypes of 22 clinical MDR
||Electrophoresis of plasmids extracted from Klebsiella
isolates from two hospitals in Urmia/Iran. Lane 1 and 2 unique profile
of plasmid band extracted from 25.6% of isolates
Electrophoresis of plasmids digested by restriction
enzymes. Lane 2: molecular size marker, Lane 1: Single digestion of
plasmid band with HincII, Lane 3: Single digestion of plasmid
band with EcoRI
Two bands obtained after restriction enzyme analysis
of plasmids. The sizes of these bands were found to be approximately 4 and 2.8
kbp (Fig. 2). The association between presence of plasmids
and resistance to antibiotics was significant (p<0.05) (Table 3).
All the plasmid containing isolates were resistant to at least ten different
Klebsiella is an opportunistic pathogen and is a causative agent
of several kinds of infections in humans.
||Relation between presence or absence of plasmid and
antibiotic resistance with Fisher`s exact statistical test. There
is meaningful relations between the presence of plasmid and resistant
to all tested antibiotics with the exception of ampicillin
It is one of the major pathogens in nurseries, intensive care units and
in hospital wards in spite of many effective antibiotics now available
(Eisen et al., 1995).
The use of broad spectrum antibiotics in hospital environments exerts
selective pressure on bacteria, results in promoting infections by multi-antibiotic
resistant isolates. Present finding showed that the most useful antibiotics
for infections caused by Klebsiella sp. were amikacin, nalidixic
acid and ciprofloxacin. Resistance to some antibiotics such as ampicillin,
gentamicin, kanamycin and nitrofurantoin showed increases in comparison
with previous studies in different countries (Rasool et al., 2003;
Talbot et al., 1980).
Isolates grouped in the main antibiotype (antibiotype 1) consisting of
eight isolates were resistant to all tested antibiotics, two isolates
grouped in antibiotype 15 were resistant to ten antibiotics and only showed
intermediate sensitivity to amikacin.
Molecular methods such as plasmid profile analysis and restriction enzyme
analysis of plasmids have recently been used for determining the bacterial
strains which cause nosocomial outbreaks. Tayfour et al. (2005)
used plasmid profiling, Restriction Endonuclease Analysis of Plasmids
(REAP) and antibiotic sensitivity tests for detection the source of resistance
in Staphylococcus aureus strains. Tolmasky et al.
(1988) reported a multiresistant Klebsiella pneumoniae
strain isolated from neonates in Argentina, harbored a 48 kbp plasmid,
with genetic determinants for resistance to amikacin and also ampicillin,
kanamycin, streptomycin and tobramycin. Gaynes et al. (1988) reported
that clinical isolates of Klebsiella at a hospital that had used
amikacin as its principal aminoglycoside demonstrated high-level resistance
to amikacin, kanamycin, gentamicin, netilmicin and tobramycin. The resistant
strains contained an identified 6.8 kbp plasmid. In the present study
plasmid DNA was detected in 25.6% of isolates, however 80% of these isolates were resistant to all tested antibiotics and 20% were resistant to 10 antibiotics and showed intermediate sensitivity to amikacin.
The prevalence of plasmids in our isolates are relatively low, therefore
it is postulated that most of the resistance genes in our isolates are
chromosomal. However all the isolates containing plasmid were resistant
to at least 10 antibiotics indicating the importance of presence of plasmid
in Multi drug resistant isolates. The relationship between presence of
certain plasmids and resistance to some antibiotics in Klebsiella sp.
has been demonstrated previously (Tolmasky et al., 1988; Gaynes
et al., 1988; Nikbin et al., 2007).
The absence of plasmids from the majority of isolates showed low typeability
power of this technique, so, using of the other molecular typing techniques
in companion with plasmid profiling and restriction enzyme analysis suggested
for further studies.
In conclusion these results remind us again that physicians should be
aware of current antimicrobial susceptibility patterns of pathogens in
their communities and design strategies to diminish non specific use of
antimicrobial drugs in the hospitals.
Research grant received from the student research committee of Urmia
University of Medical Sciences is gratefully acknowledged.