Phenotypic and Molecular Detection of OXA-48 Gene

Carbapenem-resistant Klebsiella Pneumoniae and Escherichia Coli

Isolates in Haji Adam Malik Hospital Medan, Indonesia

Mirzan Hasibuan

1,2

, R. Lia Kusumawati

2,3,4*

and Dwi Suryanto

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Jalan Bioteknologi

No. 1 Kampus USU, Medan 20155, Indonesia

University of Sumatera Utara Hospital, Jalan dr. T Mansyur No. 66 Kampus USU Medan 20154, Indonesia

Department of Microbiology, Faculty of Medicine, Universitas Sumatera Utara, Jalan Universitas No. 1 Kampus USU

Medan 20155, Indonesia

Haji Adam Malik Hospital Medan, Jl. Bunga Lau No.17, Kemenangan Tani, Medan Tuntungan, Kota Medan, Sumatera

Utara 20136, Indonesia

Keywords: Phenotypic, Molecular Detection, OXA-48, Carbapem-Resistant, Carbaenemase

Abstract: Carbapenem-resistant to Klebsiella pneumoniae and Escherichia coli, increasingly reported as a major cause

of infection in hospitals and healthcare facilities. Carbapenemase is an enzyme produced by gram-negative

bacteria that causes failure of antibiotic therapy, especially the carbapenem. The study aimed to characterize

phenotypically using Vitek 2 Compact and detect OXA-48 clinical isolates of Klebsiella pneumoniae and

Escherichia coli belonging to Carbapenem-Resistant using PCR. The results showed that all were beta-

lactamase producers, which of 12 (14.11%) were resistant to carbapenem. The phenotype distribution of

carbapenem is 10/12 (11.75%) Klebsiella pneumoniae and 2/12 (2.35%) Escherichia coli. From 12 isolates

Carbapenemase phenotypically, in which 10 (11.77%) of both bacteria bearing OXA-48 gene with the

distribution 9 (10.6%) Klebsiella pneumoniae and 1 (1.17%) Escherichia coli, respectively. The study

shows that prevalence of OXA-48 genes in North Sumatra, Indonesia in Klebsiella pneumoniae and

Escherichia coli which cause failure of therapeutic types of antibiotics Carbapenem. The only antibiotic that

is still sensitive to carbapenem-resistant bacteria based on antimicrobial susceptibility is amikacin, which

can be recommended as carbapenemases therapy.

1 INTRODUCTION

The spread of resistance to carbapenem by

Enterobacteriaceae, especially Klebsiella

pneumoniae and Escherichia coli (Maryam et.al,

2017). The emergence of bacterial resistance to the

carbapenem is a global problem that is growing

rapidly and requires urgent action for the

international scientific community (CDC, 2013).

Tropical countries such as Indonesia, infections by

Klebsiella pneumoniae and Escherichia coli are

strongly associated with health care such as the use

of health facilities that cause urinary tract infections,

as well as postoperative wound care (Raka et.al,

2006).

Carbapenem-resistant over the past decade,

healthcare settings have emerged due to the bacterial

infection of Enterobacteriaceae and are strongly

related to the ability of bacteria to produce β-

lactamase, which is capable of hydrolyzing

carbapenems (Nordmann et.al, 2012). Most of these

phenomena are related to the spread of various types

of β-lactamases. Carbapenem-hydrolysing β-

lactamase major in Enterobacteriaceae is class A

Klebsiella pneumoniae carbapenemases and class B

acquires Metallo beta-lactamases (MBLs) which is

dominated by Klebsiella pneumoniae which has

shown rapid international spread. In the D-class β-

lactamase OXA-48 despite its weak activity in

resistance but is increasingly reported to be

significant in Enterobacteriaceae. General

epidemiological observations and recent research

shows that OXA-48 producers are increasingly

being identified in various developing countries

(Nordmann et.al, 2014).

590

Hasibuan, M., Kusumawati, R. and Suryanto, D.

Phenotypic and Molecular Detection of OXA-48 Gene Carbapenem-resistant Klebsiella Pneumoniae and Escherichia Coli Isolates in Haji Adam Malik Hospital Medan, Indonesia.

DOI: 10.5220/0010079805900594

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

590-594

ISBN: 978-989-758-449-7

The increasing prevalence of carbapenem-

resistant almost all over the world provides the basis

for the importance of phenotypic characterization

and molecular detection of the Carbapenemase gene

in the Laboratory of Clinical Microbiology.

Molecular detection of the OXA-48 gene in bacteria

that produce carbapenemase enzymes is needed to

control antibiotic resistance, avoid bacterial

transmission and improve therapy management. The

aim of the study was to characterize phenotypically

and detect OXA-48 genes in isolates of Klebsiella

pneumoniae and in Escherichia coli resistant to

carbapenem in Haji Adam Malik Hospital Medan,

Indonesia.

2 METHOD

2.1 Phenotypic Characterization

Phenotypic characterization was carried out by

identification of bacterial types and sensitivity

testing of antibiotics as well as carbapenemase

phenotype using Vitek 2 Compact. The sample was

taken purely sampling based on germ pattern in the

first semester of 2015. Bacterial isolates were

obtained from routine examination of the Laboratory

of Clinical Microbiology, Installation of Diagnostic

Laboratory Haji Adam Malik Hospital Medan.

2.2 Isolation of DNA

Isolation of DNA was isolated by the freeze-thaw

cycling method before the isolate was subcultured

on Mac-Conkey Agar medium with a 24-hour

incubation period at 37°C. One colony was put into

a tube containing 20μl of aquabidest and

homogenized, then frozen for 10 minutes at 20°C,

followed by heating for 10 minutes at 90°C, hot and

frozen cycles were carried out 6 cycles. The

isolation of DNA was centrifuged for 5 minutes at a

speed of 13.000 rpm. The supernatant part is

separated, DNA purity is measured using a

nanophotometer.

2.3 Molecular Detection of OXA-48

Molecular detection using specific primers:

GCG-TGG-TTA-AGG-ATG-AAC-AC forward and

CAT-CAA-GTT-CAA-CCC-AAC-CG reverse. PCR

preparation began with DNA amplification at 25μl,

consisting of 12.5μl mastermix green go-Taq, 8.5µl

nuclease-free water, 1μl of forward and reverse

primer and 2μl bacterial DNA. Amplification is

carried out on Thermocycling reactions. PCR results

were electrophoresed and documented in UV

Reader.

3 RESULTS AND DISCUSSIONS

3.1 Phenotype

Profile of the Vitek 2 Compact antimicrobial

susceptibility from 85 clinical isolates showed that

all isolates are ESBLs, consisting of 52% of

Klebsiella pneumoniae and 48% of Escherichia coli,

respectively. The percentage profile of antimicrobial

sensitivity of the samples is presented in Table 1.

Table 1: Percentage of Antibiotic Sensitivity Tests.

Group Antibiotics

Agents

(%)

Penicillin Amoxycillin 0 100

Ampicillin 0 100

Cephalosporin Cefotaxime 0 100

Ceftriaxone 0 100

Ceftazidime 0 100

Cefepime 0 100

Monobactam Aztereonam 0 100

Beta-lactamase

Inhibitor

Amoxicillin/

Clavulanic

Acid

27 73.0

Piperacillin/

Tazobactam

43.5 56.4

Cefoperazone/

Sulbactam

63.5 36.4

Aminoglicosides Gentamycin 35.3 64.7

Amikacin 100 0

Carbapenem Ertapenem 85.9 14.1

Meropenem 85.9 14.1

Imipenem 85.9 14.1

Fluoroquinolone Ciprofloxacin 0 100

Levofloxacin 25.8 74.2

Fosfomycin Fosfomycin 86.5 13.5

Folate Pathway

Inhibitor

Cotrimoxazole 0 100

Tigecil Tigecycline 84.5 15.5

All isolates showed resistance (R) to beta-lactam

group antibiotics (penicillins, monobactams,

cephalosporins), which indicates that all isolates

carrying Extended-Spectrum Beta-Lactamases

(ESBLs). Resistance was also shown in the beta-

lactamase inhibitor group of 73% amoxicillin/

clavulanic acid, 56% piperacillin/tazobactam and

Phenotypic and Molecular Detection of OXA-48 Gene Carbapenem-resistant Klebsiella Pneumoniae and Escherichia Coli Isolates in Haji

Adam Malik Hospital Medan, Indonesia

591

36.4% cefoperazone/sulbactam, respectively.

Resistance is also shown in other classes of

antibiotics such as ciprofloxacin and cotrimoxazole.

From 85 isolate were found to be 12 (14.2%)

resistant to carbapenem (ertapenem, meropenem

imipenem), of which 2 (2.35%) Escherichia coli and

10 (11.77%) Klebsiella pneumoniae. which

indicated that the isolates are carrying ESBls and

Carbapenemases. The only antibiotic that is still

sensitive (S) to all isolates based on antimicrobial

susceptibility is amikacin.

Resistance occurs because of the genes that

encode resistant and expressed phenotypically.

Resistance occurs because of the genes that encode

resistant and expressed phenotypically. This is very

important because enzymes produced by bacteria

will express genes for resistance (Thenmozhi et.al,

2014). The reliability of the Vitek 2 Compact

System as an ESBLs and Carbapenemases detection

system was verified in comparison with another

method such as double disc synergy which is

recommended by Clinical Laboratory Standards

Institute. The characterization of the Carbapenemase

phenotype with a combination of Modified Hodge

Test (MHT) using discs and ertapenem and

meropenem is preferred. In addition, the MBL strip

E-test was also used to evaluate Metallo-β-lactamase

production (CLSI, 2013).

From the results of the Carbapenemase

phenotyping using Vitek 2 Compact, it is necessary

to proceed at the molecular level to detect ESBLs

encoding genes as well as the accuracy of both

methods. Then amplification and molecular

detection of OXA-48 gene against these 12 isolates

by using PCR.

3.2

Molecular Detection of OXA-48

Gene

Molecular detection of the OXA-48 gene showed

DNA amplification 389 bp in Klebsiella pneumoniae

and Escherichia coli (figure 1.). Based in this study

found 10/12 OXA-48 gene with the percentage of

11.77%.

Figure 1: profiles of OXA-48 gene

K- (Escherichia coli Non-Carbapenemase),

IS29 (Klebsiella pneumoniae 11474),

IS30 (Klebsiella pneumoniae 11527),

IS32 (Klebsiella pneumoniae 11583),

IS33 (Klebsiella pneumoniae 11665),

IS34 (Klebsiella pneumoniae 11417),

IS46 (Klebsiella pneumoniae 11311),

IS57 (Escherichia coli 11926),

IS58 (Klebsiella pneumoniae 11688).

From Figure 1 it can be seen that the appearance of

the DNA band pattern indicates that the bacteria has

the

OXA-48 gene. Proving that the primary can be

used OXA-48 produces 389 bp amplicon. In the

previous study, detecting the OXA-48 gene using

the same primary, where 27 of the 28 isolates of the

Motahari Hospital, Tehran, Iran (Azimi et.al, 2014).

From 12 isolates carbapenemase phenotypically,

it was found that 10 (11.77%) Klebsiella

pneumoniae and 2 (2.35%) of Escherichia coli

isolates showed carbapenemase, isolates containing

OXA-48 gene were 10 (11.77%) which are

presented in table 2. Another 2.35% possibility

contains other genes that cause carbapenem-

resistance.

Table 2: Percentage of ESBLs, Carbapenemases

phenotype and OXA-48 gene.

Bacterial

species

Phenotype OXA-48

gene

ESBLs Carbapenemase

K.pneumoniae 44 (52%) 10(11.77%) 9(10.6%)

E.coli 41 (48%) 2(2.35%) 1(1.17%)

total : 85(100%) 12 (14.12%) 10(11.77%)

The results of this study showed the spread of OXA-

48 gene in Indonesia. In this study, the OXA-48

gene was not only found in Klebsiella pneumoniae,

but was also found in the Escherichia coli. In 2

phenotypic Carbapenemase isolates, not found the

blaOXA-48 gene. There may be other genes such as

IMP-1, NDM-1 and other types of carbapenemase

genes. Molecular detect of ESBLs genes by

Karuniawati et.al (2013) in RSPN Cipto

Mangunkusumo Jakarta, Indonesia. From the 61

Gram-negative bacteria-producing carbapenemases

in phenotype, did not have the OXA-48 type gene.

However, the gene encoded carbapenemases such as

IMP-1 and NDM-1.

The presentation of carbapenem-resistant in

Enterobacteriaceae poses a major problem for health

services, for example the limited choice of antibiotic

therapy. As a bacterium producing Carbapenemase,

these bacteria are not only resistant to carbapenem

but almost resistant to all beta-lactam groups, except

monobactam (aztreonam) for MBL and other

compounds such as OXA-48 (Jacoby et.al, 2004). In

this study, it was found that all isolates were even

resistant to aztreonam. In addition, the extended

spectrum β-lactamase (ESBL) is strongly associated

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

592

with carbapenem resistance in Enterobacteriaceae,

this is due to the female bacteria producing AmpC β-

lactamase or loss of porin in the bacterial cell wall

(Woodford, 2007).

This gene OXA-48 is also known as the

carbapenemase coding gene in gram-negative

bacteria. The first identified OXA-48 gene

manufacturer was derived from the Klebsiella

pneumoniae strain isolated in Turkey in 2003. Since

then, manufacturers of

bla

OXA-48 have been widely

reported in Turkey as a source of bacteria that cause

nosocomial infections (Nordmann et.al, 2004). The

worldwide distribution of OXA-48 manufacturers

now covers countries in Europe, Africa, America,

and even Asia. The spread of multi-resistant

pathogens worldwide has been linked to a variety of

epidemiological factors including international

patient transfer originating from endemic areas

(Girmenia et.al, 2016). The argument about the

spread of Klebsiella pneumoniae as a type of

bacteria that causes carbapenemase is considered a

high-risk organism (Munoz-Price et.al, 2013).

The threat of antimicrobial resistance has been

recognized by World Health Organization, where

this threat involves and requires all actions related to

the agency and society as a whole (WHO, 2013).

Clone expansion is a major driver of the spread of

carbapenemase in gram-negative bacteria, especially

in Enterobacteriaceae. In addition, carbapenemase

transmission as a clonal lineage of

Enterobacteriaceae (CPE) is stable in the defense of

the carbapenemase coding gene. Horizontal transfer

of this type of gene is very likely to occur through

the moving genetic element, the plasmid (Kitchel

et.al, 2009).

Study on the detection of OXA-48 genes is still

rare in Indonesia, especially in North Sumatera. This

study shows the presence of the OXA-48 gene as a

cause of Carbapenemase in Klebsiella penumoniae

and Escherichia coli. This study also provides an

overview of the prevalence and incidence of

resistance to carbapenem, for this reason the

awareness of all hospitals is the importance of

controlling resistance in the present and avoiding the

transmission of resistant bacteria in health facilities.

4 CONCLUSIONS

The prevalence of carbapenemase has been shown in

this study, found of the phenotype 12 (14.12%) and

molecularly detected 10 (11.77%) OXA-48 genes.

However, there were 2 other isolates that were not

found in the OXA gene, possibly another gene that

causes carbapenemase.

ACKNOWLEDGEMENTS

The author would to thanks Director of Haji Adam

Malik General Hospital Medan, Indonesia,

especially for Clinical Microbiology Laboratory.

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