Medical Journal of Babylon-Vol. 12- No.

1 -2015 ٢٠١٥ - ‫اﻟﻌﺪد اﻷول‬-‫ اﻟﻤﺠﻠﺪ اﻟﺜﺎﻧﻲ ﻋﺸﺮ‬-‫ﻣﺠﻠﺔ ﺑﺎﺑﻞ اﻟﻄﺒﯿﺔ‬

Molecular Detection of CTX-M Genes in Klebsiella pneumoniae Isolated

from Different Clinical Samples in Baghdad City

Ahmed Salim Mohammed

College of Health and Medical Technology, Baghdad

Received 15 December 2014 Accepted 28 January 2015

Abstract
CTX-M extended-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae have been reported to be
an important nosocomial infections. A total of 50 K. pneumonia isolates were isolated from different clinical
samples in some public hospitals in Baghdad city during the period from October to December 2013.
Bacterial identification was done using conventional cultural & chemical methods &and VITEK 2 cards (GN)
for identification, while the antimicrobial drug susceptibility of K. pneumoniae was performed by disk
diffusion test and the minimum inhibitory concentration (MIC) testing was performed using VITEK 2
automated system (bioMérieux, France). ESBL production was phenotypically detected by double disk
synergy test according to the Clinical and Laboratory Standards Institute(CLSI) guidelines. The presence of
bla-gene encoded CTX-M was detected by conventional PCR technique.
Out of 50 K. Pneumonia isolates,13 (26%) were ESBL producer by CDT, the minimum inhibatory
concentration (MIC) of different antibiotics was performed on these 13(26%) isolates using VITEK2 AST-
GN30 showed that 13 (100%) isolates were Ceftazidime,Ceftraiaxone and Cefepime resistant with MIC ≥16-
≥64 µg/ml,and 8(61.53%) of ESBL producing isolates were carbapenem sensitive 8 (61.53%) with MIC
<=0.25 µg/ml. PCR assay revealed that 4 (30.76%) of the ESBL producing isolates harbored blaCTX-M
gene.
Extended spectrum beta lactamase mediated resistance in K. pneumonia is a cause for concern in the therapy
of critically ill patients. The ESBL producing K. pneumoniae isolates were more resistant to various
antimicrobial agents. This suggests that ESBL producing isolates in hospitals may cause serious infections
that illustrated when these strains were responsible for a nosocomial outbreak.The findings strongly suggest
that there is a need to track the detection of ESBL producers and that judicious use of carbapenems is
necessary to prevent the further spread of these organisms. The prevalence of multi-drug resistant K.
pneumoniae isolates especially ESBL producing bacteria was increased in Baghdad city .Phenotypic and
molecular characterization of ESBL provide information about the prevalence of ESBL producing K.
pneumoniae in Baghdad. The blaCTX-M was one of the predominant ESBL genes in K. pneumoniae in this
study.
Key words: Klebsiella pneumonia, ESBL, bla genes

‫ ﻓﻲ ﺑﻜﺘﺮﯾﺎ اﻟﻜﻠﺒﺴﯿﻼ اﻟﺮﺋﻮﯾﺔ واﻟﻤﻌﺰوﻟﺔ ﻣﻦ ﻣﺨﺘﻠﻒ اﻟﻌﯿﻨﺎت‬CTX-M ‫اﻟﻜﺸﻒ اﻟﺠﺰﯾﺌﻲ ﻋﻦ ﺟﯿﻨﺎت‬

‫اﻟﺴﺮﯾﺮﯾﺔ ﻓﻲ ﻣﺪﯾﻨﺔ ﺑﻐﺪاد‬
‫اﻟﺧﻼﺻﺔ‬
‫ ﻣﺛﺑﺗﻪ ﺑﺎﻧﻬﺎ ﻣن اﻟﻣﺳﺑﺑﺎت اﻟﻣﻬﻣﺔ ﻟﻼﺻﺎﺑﺎت واﻻﻟﺗﻬﺎﺑﺎت اﻟﻣﺧﺗﻠﻔﺔ ﻓﻲ‬CTX- M ‫ﺗﻌﺗﺑر ﺑﻛﺗرﯾﺎ اﻟﻛﻠﺑﺳﯾﻼ اﻟرﺋوﯾﺔ اﻟﻣﻧﺗﺟﺔ ﻻﻧزﯾﻣﺎت‬
‫ ﺑﻛﺗرﯾﺎ اﻟزواﺋف اﻟزﻧﺟﺎرﯾﺔ ﻟﻬﺎ ﻗﺎﺑﻠﯾﺔ ﻋﺎﻟﯾﺔ ﻋﻠﻰ ﻣﻘﺎوﻣﺔ اﻟﻌدﯾد ﻣن اﻟﻣﺿﺎدات اﻟﺣﯾﺎﺗﯾﺔ اﻟﻣﺳﺗﺧدﻣﺔ ﺣﺎﻟﯾﺎ ﻣن ﺧﻼل ﻣﯾﻛﺎﻧﯾﺎت ﻣﺧﺗﻠﻔﺔ‬.‫اﻟﻣﺳﺗﺷﻔﯾﺎت‬
‫ ﻋزﻟﺔ ﻣن ﺑﻛﺗرﯾﺎ‬50 ‫ ﺗم ﺟﻣﻊ‬. Extended spectrum B-Lactamase ‫ﻣﻧﻬﺎ داﺧﻠﯾﺔ او ﻣﻛﺗﺳﺑﺔ واﻫم اﻟﻣﯾﻛﺎﻧﯾﻛﯾﺎت ﻫو اﻧﺗﺎج اﻧزﯾﻣﺎت‬
‫اﻟﻛﻠﺑﺳﯾﻼ اﻟرﺋوﯾﺔ ﻣن ﻣﺧﺗﻠف اﻟﻌﯾﻧﺎت اﻟﺳرﯾرﯾﺔ ﻣن ﺑﻌض اﻟﻣﺳﺗﺷﻔﯾﺎت واﻟﻣﺧﺗﺑرات اﻟﺣﻛوﻣﯾﺔ ﻓﻲ ﻣﺣﺎﻓظﺔ ﺑﻐداد ﻟﻠﻔﺗرة ﻣن اب وﻟﻐﺎﯾﺔ ﻛﺎﻧون اﻻول‬
‫ ﺗم ﺗﺷﺧﯾص اﻟﺑﻛﺗرﯾﺎ ﺑﺎﺳﺗﺧدام ﻣﺧﺗﻠف اﻟطرق ﺳواء ﻛﺎﻧت ﻓﺣوص ﻛﯾﻣﯾﺎﺋﯾﺔ او ﻋن طرﯾق ﺗﺷﺧﯾص اﻟﺑﻛﺗرﯾﺎ ﻓﻲ اوﺳﺎط زرﻋﯾﺔ‬.٢٠١٣ ‫ﻟﻌﺎم‬
‫ وﺗم ﻗﯾﺎس اﻟﺣﺳﺎﺳﯾﺔ اﻟدواﺋﯾﺔ ﻟﻠﻌزﻻت اﻟﺑﻛﺗﯾرﯾﺔ ﺑﺎﺳﺗﺧدام ﻧﻔس اﻟﺟﻬﺎز‬VITEK2 ‫ﻛذﻟك ﺗم ﺗﺷﺧﯾص اﻟﻌزﻻت اﻟﺑﻛﺗﯾرﯾﺔ ﺑﺎﺳﺗﺧدام ﺟﻬﺎز‬,‫ﻣﺧﺗﻠﻔﺔ‬
‫وﻋدد اﻟﻌزﻻت اﻟﺑﻛﺗﯾرﯾﺔ اﻟﻣﻧﺗﺟﺔ‬ 100% ‫ﻧﺳﺑﺔ اﻧﺗﺷﺎر اﻟﻌزﻻت اﻟﻣﻘﺎوﻣﺔ ﻟﻌﻘﺎرات اﻟﺳﯾﻔﺎﻟوﺳﺑورﯾن ﻫﻲ‬:‫وﻛذﻟك ﺗم اﻟﺗوﺻل ﻟﻠﻧﺗﺎﺋﺞ اﻟﺗﺎﻟﯾﺔ‬
‫ﻟﻠﻌزﻻت اﻟﺛﻼﺛﺔ ﻋﺷر اﻟﻣﻘﺎوﻣﻪ اﻟﻣﻧﺗﺟﺔ ﻣظﻬرﯾﺎ‬PCR ‫ ﺗم اﺳﺗﺧدام ﺗﻘﻧﯾﻪ ال‬.26% ‫ وﺑﻧﺳﺑﺔ‬13 ‫ﺑﺎﺳﺗﺧدام اﻟطرق اﻟﻣظﻬرﯾﺔ ﻫو‬ESBL‫ﻻﻧزﯾﻣﺎت‬

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‫واظﻬرت اﻟﻧﺗﺎﺋﺞ ان‬CTX-M‫ واﻟﻣﺷﻔرة ﻻﻧزﯾﻣﺎت‬blagenes ‫ ﻟﻠﻛﺷف ﻋن وﺟود اﻟﺟﯾﻧﺎت اﻟﺧﺎﺻﻪ ﺑﻣﻘﺎوﻣﻪ اﻟﻣﺿﺎدات اﻟﺣﯾﺎﺗﯾﺔ‬ESBL ‫ﻻﻧزﯾﻣﺎت‬
.blaCTX-M ‫ ﻛﺎﻧت ﺗﺣوي ﺟﯾﻧﺎت‬ESBL ‫( ﻣن اﻟﻌزﻻت اﻟﻣﻧﺗﺟﺔ ﻻﻧزﯾﻣﺎت‬%٣٠.٧٦) ‫ ﻋزﻻت‬٤
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Introduction based on their amino acid sequence

K lebsiella pneumonia is gram identities. Group I includes CTX-M-1, -3,

negative bacilli, possessing non- -10 to -12, -15, -22, -23, -28, -29, and -30.
motile, facultative anaerobic Group II includes CTX-M-2, -4 to -7,
bacteria. The organisms are usually 3- and-20 and Toho-1. Group III includes
6μm in length and up to 1.0 μm in width. CTX-M-8. Group IV includes CTX-M-9,
The encapsulated strains of Klebsiella are -13, -14, -16 to -19, -21, and -27 and
known to produce mucoid colonies (1). Toho-2. Finally group V includes CTX-
Klebsiella pneumoniae is an important M-25 and -26. The members of these
pathogen that causes urinary tract groups exhibit greater than 94% amino
infections (UTIs), pneumonia, and intra- acid identity within the group and about
abdominal infections in hospitalized 90% amino acid identity between groups
immunocompromised patients with severe (10).
underlying diseases (2). It has been The CTX-M enzymes have been detected
estimated that Klebsiella spp. cause 5-7% in a many Enterobacteriaceae species,
of the total bacterial nosocomial from different geographical regions.
infections in the world (3). Beta-Lactams However, the CTX-M variants are mostly
are the most widely used antibiotics in detected in E. coli, S. typhimurium, K.
clinical medicine and resistance to beta- pneumoniae and Proteus mirabilis (11).
lactams may become a severe threat Infection with the ESBL producing
because they have low toxicity and are organisms is associated with higher rates
used to treat a broad range of infections of mortality, morbidity, and health care
(4). Cephalosporins, fluoroquinolones, costs, which is at least partly due to the
aminoglycosides and carbapenems are lack of proper screening recommend-
effective for treating infections caused by dations. Nosocomial infection involving
Klebsiella pneumoniae (5, 6). multi-drug resistant K. pneumoniae is a
Resistance to extended spectrum growing problem worldwide (12).
cephalosporins can occur in K. The present study was designed for
pneumoniae via the production of phenotypic detection of CTX-M type
extended spectrum Beta-lactamases ESBLs and molecular detection of genes
(ESBLs) that are capable of hydrolyzing encoding CTX-M-β-lactamases in ESBL-
the oxyiminocephalosporins and producing strains of K. pneumoniae
monobactams (7, 8). Although, today isolated from clinical specimens collected
hundreds variant of ESBLs have been in this study.
described but the most common of them
are derivatives of TEM or SHV enzymes Materials and Methods
also, In recent years a new family of Bacterial Isolates
plasmid mediated CTX-M extended Fifty isolates of Klebsiella pneumonia
spectrum β-lactamases (ESBLs) called were isolated from different clinical
CTX-M has arisen with increasing samples in Baghdad/Iraq during the
frequency from Europe, Africa, Asia, period from October to December 2013.
South America and North America. These The Klebsiella isolates (50 isolates) were
ESBLs were named CTX-M type Beta- as follows: burn (17), ear (3), sputum (4),
lactamases, owing to their high activity wound (2), urine (19), vagina (2), blood
against cefotaxime (9). (3). Clinical samples were collected from
According to a recent review and new teaching laboratories of medical city, Al-
data within GenBank, CTX-M-β- Yarmouk Hospital, in addition to some
lactamases can be divided into five groups private laboratories. Bacteria were

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cultured on MacConkey and Nutrient agar suspension and incubated for 5 minutes at
in aerobic condition at 42 C for 24-48 h, 55 oC.
Then identified by conventional 3- About 2 ml of 5M NaCl solution
biochemical tests and by using of VITEK was added to the lysate, mixed thoroughly
2 Automated system using (GN) cards. by inversion and let to be cooled to
Antibiotic Susceptibility Testing 37 oC.Then 5 ml of (phenol: chloroform:
All K. pneumoniae isolates were cultures isoamylalcohol) (25: 24: 1 v/v) was added
on MacConkey agar (selective and to the lysate and mixed by inversion for
differential media). The antimicrobial 30 minutes at 25 o C and the spun by
susceptibility of these isolates was centrifuge 4500 rpm for 10 minutes.
achieved by disk diffusion and VITEK 2 4- The aqueous phase was
system according to CLSI (13). The MIC transferred to a fresh tube, which contain
for phenotypically ESBL producing the nucleic acid then isopropanol (0.6
isolates was obtained. volume) was added to the extract and
Identification of ESBL Producing mixed by inversion, after 3 minutes DNA
Isolates spooled on to a sealed pasture pipette.
Combined disk test (CDT) 5- The DNA rinsed in 5 ml of 70%
A disk of ceftazidime (30μg) alone and a ethanol, air dried, and dissolved in 300 l
disk of ceftazidime + clavulanic acid TE buffer, and then DNA extract was kept
(30μg/10μg) were placed independently, at -20 oC until use.
30mm apart, on a lawn culture of 0.5 Mc- Preparation of primers suspension
Farland opacity of the test isolate on The DNA primers were resuspended by
Mueller Hinton Agar (MHA) plate and dissolving the lyophilized primers after
incubated for 18-24 hours at 35°C. An spinning down with TE buffer depending
increase of ≥5 mm zone of inhibition on IDT/USA instructions as stock
diameter around the ceftazidime/ suspension. Working primer tube was
clavulanic acid in comparison to prepared by diluted with TE buffer. The
ceftazidime confirmed ESBL production final picomoles depended on the
(13). procedure of each primer.
Molecular detection of blaCTX-M Detection of CTX-M genes by PCR
genes The CTX-M genes were detected for the
Plasmid DNA Extraction phenotypically resistant isolates by using
DNA preparation from bacterial cells was primers targeting blaCTX-M gene. The
performed by salting out method with PCR amplification mixture has been
some modification as following: prepared according to the manufacturer's
1- Bacterial cell of 50 ml culture instructions (Intron, Korea).
were precipitated by centrifugation (1000
rpm for 10 minutes). Rewashed 3 times in A- Primers:
TE buffer, Then the pellet was The primers used to amplify the genes
resuspended in 5 ml TE buffer. encoding the CTX-M enzymes are listed
2- A volume of 600 l of 25% SDS in Table -1 below:
was added, mixed by inversion to the cell

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Table 1 : primers sequences for detection of blaCTX-M genes

Gene Primer sequence (5ʹ-3ʹ) Size Ref.
of product

blaCTX-M F CGCTTTGCGATGTGCAG 550 bp (14)

blaCTX-M R ACCGCGATATCGTTGGT

B-The reaction mixture: following (Table-2). and The PCR

Amplification of DNA was carried out in product was detected using agarose gel
a final volume of 20μl containing the electrophoresis

Table 2: Contents of the reaction mixture

No. Contents of reaction mixture Volume
1. 2X PCR ImaxIImaster mix 4μl
2. Upstream primer 1μl
3. Downstream primer 1μl
4. DNA template 5μl
5. Nuclease free water 9μl
Total volume 20 μl

Results ceftriaxone and cefepime The resistance

Out of the 50 K. pneumoniae isolates profile of ESBL producing isolates against
studied, only 13(26%) appear to be carbapenems was different , Five ESBL
phenotypically ESBL producer by using producing K. pneumoniae isolates were
Combined Disk Test (CDT). (Table-3, resistant imipenem and meropenem with
Figure-1). MIC ≥16 µg/ml, while the other eight
The antibiotic susceptibility test was done isolates were sensitive to carbapenems
for all isolates K. pneumoniae isolates by with MIC <=0.25 µg/ml.
disk diffusion method .In present study, The study showed that 10 (76.9%) of
the MIC of 10 antibiotics listed in Table ESBL producing isolates were resistant to
(4) was done by VITEK2-Compact using ciprofloxacin MIC ≥4 µg/ml, while only 5
AST-GN30 for testing the antibiotic (38.4%) were resistant to levofloxacin.
susceptibility for the ESBL producing K. The results showed that 12 (92.3%) of
pneumoniae isolates (no.13) and the MIC ESBL producing K. pneumonia were
values were interpreted according to the resistant to aminoglycosides used in this
CLSI 2012 (11). study (Gentamicin and Tobramycin).
The ESBL producing K. Pneumoniae in
this study (no. 13) differ in the level of
resistance to different antibiotics including
the cephalosporins as showed in Table (4).
All of the ESBL isolates showed MIC ≥16
for ceftazidime and ≥64 µg/ml for both

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Table 3: Prevalence of ESBL producing K. pneumoniae isolates

Total number of Positive for ESBLs

isolates Numbers %
n=50 13 26%

Figure 1: Positive Phenotypic confirmatory test (combination disk method) for detection
of ESBL, Muller – Hinton agar plate showing an isolate Klebsiella pneumoniae resistant
to ceftazidime (CAZ) (30μg) and along with an increase zone of inhibition around
ceftazidime clavulanic acid (CZC) (30ug/10).

Most of the ESBL producing K. resistant to Meropenem MIC ≥16& these

pneumoniae isolates were recovered from isolates were found to be multi drug
urine (Table- 4). resistant (MDR) to the 10 antibiotics
All the isolates are resistant to Imipenem (Table- 4).
MIC ≥16, while only 4 isolates are

Table 4: Antibiotic susceptibility of ESBL producing K. pneumoniae isolates

MIC (µg/ml) of selected antibiotics determined by VITEK 2 system

Isolate Specimen IMP CIP GM CAZ AMC CRO TOB MER FEP LEVO

k1 Burn R R R R R R R R R R

>=16 >=4 >=16 >=64 >=32 >=64 >=16 >=16 >=64 >=8

K2 Burn S R R R R R R S R S

<=0.2 >=4 >=16 32 >=32 >=64 >=16 <=0.25 >=64 1

5

K3 Burn S R R R S R R S R S

0.5 >=4 >=16 >=64 1 >=64 >=16 <=0.25 >=64 1

K4 Burn R R R R R R R R R R

>=16 >=4 >=16 >=64 >=32 >=64 >=16 >=16 >=64 >=8

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K5 Sputum S R R R R R R S R S

<=0.2 >=4 >=16 32 >=32 >=64 >=16 <=0.25 >=64 1

5

K6 Sputum R R R R R R R R R R

>=16 >=4 >=16 >=64 >=32 >=64 >=16 >=16 >=64 >=8

K7 Urine R R R R R R R R R R

>=16 >=4 >=16 >=64 >=32 >=64 >=16 >=16 >=64 >=8

K8 Urine S R R R R R R S R S

<=0.2 >=4 >=16 32 >=32 >=64 >=16 <=0.25 >=64 1

5

K9 Urine S S R R R R R S R S

<=0.2 <=0.25 >=16 16 >=32 >=64 >=16 <=0.25 >=64 1

5

K10 Urine S R R R R R R S R S

<=0.2 >=4 >=16 16 16 >=64 >=16 <=0.25 >=64 1

5

K11 Urine S S S R R R S S R S

0.5 0.5 <=1 16 16 >=64 <=1 <=0.25 >=64 1

K12 Urine R R R R R R R R R R

>=16 >=4 >=16 >=64 >=32 >=64 >=16 >=16 >=64 >=8

K13 Urine S S R R S R R S R S

<=0.2 <=0.25 >=16 16 1 >=64 >=16 <=0.25 >=64 1

5

Abbreviation:IPM, imipenem; CIP, ciprofloxacin; GM, gentamicin; CAZ, ceftazidime; AMC, amoxicillin-
clavulanic acid; CRO, ceftriaxone ;TOB, tobramycin; MEM, meropenem; FEP, cefepime; LEV, levofloxacin

All Klebsiella pneumoniae isolates that isolates. The distribution of plasmid

were resistant to ceftazidime and mediated CTX-M genes within study
phenotypically ESBL producers (n=13) isolates are shown in Figure (2). The
were further investigated for the presence plasmidic blaCTX-M was detected in 4
of or plasmid mediated blaCTX-M using (30.76%) isolates, these are K1, K2, K4
specific primer. The presence of CTX-M and K6 respectively. These results
genes was detected by conventional PCR achieved using specific blaCTX-M gene
technique. primers.
PCR analysis for CTX genes was
accomplished for the 13 ESBL producer

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Figure 2: Ethidium bromide stained agarose gel showing PCR amplification products with
blaCTX-M gene (550 bp) primers for K. pneumoniae extracted DNA.
M: 100 bp standard size reference marker. Lane 1: K1 shows positive result with blaCTX-M gene
Lane 2: K2 shows positive result with blaCTX-M gene. Lane 3: K3 shows negative result with
blaCTX-M gene. Lane 4: K4 shows positive result with blaCTX-M gene. Lane 5: K5 shows
negative result with blaCTX-M gene. Lane 6: K6 shows positive result with blaCTX-M gene.

Discussion obtained in this study clarified that the K.

Klebsiella pneumoniais common pathogen pneumonia isolates showed high resistance
causing nosocomial infection.In present to most common antibiotics of β-lactams,
study, ESBL producing isolates shows aminoglycosides, fluroquinolones were
high level of resistance to all β-lactam suspected to be highly producers of
antibiotics including β-lactamase inhibitor, ESBLs; therefore, all K.pneumoniae
aminoglycosides and quinolones. isolates tested phenotypically for ESBL
Resistance to aminoglycosides was present production. The isolates that produce
in most ESBL producing isolates (13). ESBLs were 13 (26%) and they were more
According to various studies ESBL frequently among urinary tract infections.
production ranged from 10% to 65%. In A study done by Al-Muhanna showed
Present study, ESBL productions were that Isolates that produce ESBLs were
26% in isolates of Klebsiella pneumoniae predominant more frequently among burns
using CDT (confirmative test). ESBL infections in 11 (42.3%) isolates followed
positive isolates leads to serious by 7 (26.9%) of UTI. (16).
therapeutic failure because they carry Ceftazidime is a third generation
multidrug resistant genes and the cephalosporin used frequently for the
imipenem is the drug of choice for treatment of infections caused by K.
treatment (14). pneumoniae. However, the resistance to
Currently, no standardized method for ceftazidime is increasing at an alarming
ESBL detection has been proposed and rate, complicating the clinical
despite PCR being highly accurate and management of patients infected with such
reliable, its accessibility is often limited to isolates. In this study, a high level of
reference laboratories (15). resistance to ceftazidime was observed
The results of antibiotic sensitivity among the K. pneumoniae isolates

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recovered from different clinical samples Although the carbapenem was the drug of
as showed in Table (4) . This result was choice for ESBL producing K.
close to the results obtained by Al- pneumoniae isolates, the emerging of
Muhannak (2010) for ceftazidime 89.8% ESBL producing bacteria poses a threat to
(17). antibiotic treatment program in Baghdad
In this study, ESBL producing isolates hospitals.
were significantly more resistant to all The blaCTX-M was the predominant
antibiotics tested as compared to non- among the ESBL genes of K. pneumoniae
ESBL producing isolates. other studies in this study. The blaCTX-M genes are
have reported on cross resistance to encoded by a plasmid and this resistance
aminoglycosides, fluoroquinoloes, and mechanism can cause nosocomial
trimethoprim in ESBL producing outbreaks.
organisms (18). Mechanisms of co-
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