Advances in Biomolecular Medicine

Proceedings of the 4th BIBMC (Bandung International Biomolecular Medicine Conference) 2016 and the 2nd ACMM (ASEAN Congress on Medical Biotechnology and Molecular Biosciences), October 4-6, 2016, Bandung, West Java, Indonesia

Robert Hofstra, Noriyuki Koibuchi, Suthat Fucharoen

• Publisher: CRC Press
• Year: 2017
• Print ISBN: 9781138631779
• eBook ISBN: 9781351804172
• Topic: Biological Sciences
• Subtopic: Biotechnology in Medicine
• Index: Biological Sciences

Advances in Biomolecular Medicine contains the selected papers presented at the 4th BIBMC (Bandung International Biomolecular Medicine Conference) and the 2nd ACMM (ASEAN Congress on Medical Biotechnology and Molecular Biosciences), hosted by the Faculty of Medicine, Padjadjaran University, Bandung, West Java, Indonesia, 4-6 October 2016. In line with the United Nations Sustainable Development Goals, the theme of the joint scientific meeting is 'Medical innovation & translational research to ensure healthy lives & promote well-being for all at all ages'.

Authors include scientists, academics, practitioners, regulators and other key individuals with expertise and experience relevant to biomolecular medicine, medical biotechnology and molecular biosciences. Topics of the papers cover various aspects of infection, oncology, tuberculosis, genetics, thalassemia, nutrition, cardiovascular, wound healing and endocrinology. This book is essential reading for academics, scientist, practitioners and regulators involved in the area of biomolecular medicine, medical biotechnology and molecular biosciences.

Molecular detection of DHA-1 AmpC beta-lactamase gene in Enterobacteriaceae clinical isolates in Indonesia

B. Diela

Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

S. Sudigdoadi,A.I. Cahyadi,B.A.P. Wilopo & I.M.W. Dewi

Department of Microbiology and Parasitology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

C.B. Kartasasmita

Department of Pediatrics, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia Dr. Hasan Sadikin Hospital, Bandung, Indonesia

ABSTRACT: Dhahran (DHA)-1 is an inducible plasmid-mediated AmpC beta-lactamase gene, which was reported as the most frequent cause of escalating morbidity and mortality rate among patients with infectious diseases. The aim of this study was to determine the prevalence of bla_DHA-1 gene in Enterobacteriaceae clinical isolates in Indonesia. A total of 85 Enterobacteriaceae clinical isolates, including E. coli, Klebsiella. pneumoniae, E. cloacae, K. oxytoca, E. aerogenes, and Proteus mirabilis, were included in this study. The samples were collected from several private hospitals and clinical laboratories in Bandung, Indonesia, during a period between July 2014 and April 2015. The molecular detection of bla_DHA-1 gene was carried out by PCR assay. We found that 32.9% of Enterobacteriaceae clinical isolates carried bla_DHA-1 gene, which was predominantly found in K. pneumoniae (75%) followed by K. oxytoca (50%), E. cloacae (28.6%), and E.coli (14%). These results revealed a higher frequency of bla_DHA-1 gene detected in Enterobacteriaceae clinical isolates in Indonesia compared with those in surveillance studies conducted worldwide. To our knowledge, this is the first molecular epidemiology report from Indonesia that showed the high prevalence of bla_DHA-1 gene in Enterobacteriaceae clinical isolates.

Keywords: AmpC Beta-Lactamase; DHA-1; Enterobacteriaceae; PCR; Indonesia

1 INTRODUCTION

Inadequate regulation of antibiotic therapy for many infectious diseases in most Asian countries, including Indonesia, may increase resistance spectrum of gram-negative bacteria to beta-lactam antibiotics.⁴ With the increasing use of beta-lactam antibiotics and various inhibitor combinations, such as amoxicillin-clavulanic acid or sulbactam, production of AmpC beta-lactamase enzyme by gram-negative bacteria have emerged.⁴ This enzyme can hydrolyze and inactivate the third generation of beta-lactam antibiotics, including oxyimino-cephalosporins (cefotaxime, ceftazidime, and ceftriaxone), monobactam (aztreonam), and cephamycins (cefoxitin and cefotetan).⁴ AmpC beta-lactamase enzyme has been mainly detected in Enterobacteriaceae family of gram-negative bacteria and frequently causes severe infection cases in hospitalized patients.⁴ This enzyme is poorly inhibited by many common beta-lactamase inhibitors, such as clavulanic acid and sulbactam, but can only be inhibited by AmpC inhibitors, such as cloxacillin or 3-aminophenylboronic acid.^2,3

Detection of AmpC beta-lactamase enzyme is important to improve the clinical treatment for patients with infection due to Enterobacteriaceae. Recently, several phenotypic tests for detection of this enzyme are being developed with varying degrees of success, such as AmpC Disk Test, 3-Aminophenylboronic Acid Disk Test, and Three-Dimensional Test.^5,6 However, there is no specific phenotypic test to confirm the presence of AmpC beta-lactamases in clinical isolates which has been validated by Clinical Laboratory Standards Institute (CLSI).⁴ Therefore, there is a need to conduct an examination using PCR (Polymerase Chain Reaction) as the gold standard method to detect the presence of AmpC beta-lactamase gene in Enterobacteriaceae clinical isolates.^2,7

The AmpC beta-lactamase gene (bla_AmpC) consists of 6 families: ACC, FOX, MOX, DHA, CIT and EBC. These are encoded either in chromosome or plasmid.^3,8 The expression of bla_AmpC chromosomal gene can be triggered by the presence of beta-lactam antibiotics, especially cefoxitin.^3,7 The plasmid-mediated bla_AmpC gene is derived from the bla_AmpC chromosomal gene or can be transferred horizontally between plasmids of Enterobacteriaceae.^3,5,9 DHA-1 is a subfamily of DHA, an inducible plasmid-mediated bla_AmpC gene, which has been reported as the most frequent cause of escalating morbidity and mortality rate among infected patients compared with other families of plasmid-mediated bla_AmpC gene.^10,12 Bla_DHA-1 gene was originated from the chromosomal bla_AmpC gene of Morganella morganii, which the expression can be induced by the presence of beta-lactam antibiotics. Thus, the increasing use of beta-lactam antibiotics in many of infection cases may induce the expression of bla_DHA-1 gene among Enterobacteriaceae clinical isolates.^3,10,11

At present, there has not been any molecular epidemiological data in Indonesia that report the prevalence of bla_DHA-1 gene in Enterobacteriaceae. Therefore, this study was designed to detect the presence of bla_DHA-1 gene in Enterobacteriaceae clinical isolates in Indonesia by using PCR assay. The results will make health care providers to be more careful in administering antimicrobial therapy for infected patients to decrease the occurrence of antimicrobial resistance and the expansion of resistance spectrum of Enterobacteriaceae to beta-lactam antibiotics.

2 MATERIALS AND METHODS

2.1 Bacterial isolates

Ninety two Enterobacteriaceae clinical isolates were collected from several private hospitals and clinical laboratories in Bandung, Indonesia, during a period between Juli 2014 and April 2015.

These isolates were taken from patient’s specimens, including blood, urine, abdominal pus, vaginal swab, etc. The organisms were grown on nutrient agar plates and biochemical tests were carried out to identify the bacterial species of the Enterobacteriaceae clinical isolates. Eighty five Enterobacteriaceae clinical isolates were included in this study, including Escherichia coli (n = 50), Klebsiella pneumoniae (n = 24), E. cloacae (n = 7), K. oxytoca (n = 2), E. aerogenes (n = 1), and Proteus mirabilis (n = 1). Seven unknown or non-specific isolates were, such as Klebsiella sp., Serratia sp., or rod-shaped gram-negative bacteria, were excluded from the study.

2.2 Plasmid DNA isolation

Plasmid DNA from Enterobacteriaceae clinical isolates were isolated using the Xprep Plasmid DNA Mini Kit (PhileKorea Technology Inc., Seoul, Korea) according to the manufacturer’s instructions.

2.3 PCR amplification of bla_DHA-1 gene

The oligonucleotide primers used for PCR assay were DHAM-forward (F) (5’-AAC TTT CAC AGG TGT GCT GGG T-3’) and DHAM-reverse (R) (5’-CCG TAC GCA TAC TGG CTT TGC-3’), which were designed by Pérez-Pérez FJ⁴ and synthesized by Integrated DNA Technology, Inc., Lowa, USA. Reactions were carried out in a MasterCycler Gradient™ (Eppendorf Inc., Hamburg, Germany) in 12.5 μL of mixtures containing 6.25 μL of DreamTaq Green PCR Master Mix™ (Thermo Fisher Scientific Inc., MA, USA), which consists of DreamTaq DNA polymerase, 2X DreamTaq Green Buffer, dNTPs, and 4 mM MgCl₂; 0.125 μL of 10 μM DHAM-F Primer; 0.125 μL of 10 μM DHAM-R Primer; 5.5 μL of nuclease-free water; and 0.5 μL of isolated plasmid DNA.

The PCR condition used in this study was; 1 cycle of initial denaturation at 95°C for 2 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 1 minute and t72°C for 1 minute and then a final extension at 72°C for 10 minutes.

2.4 Electrophoresis

12 μL of PCR products were resolved on 1% agarose gels, which was made by 3 mg of TopVision™ Agarose Gel (Thermo Fisher Scientific Inc., MA, USA). For negative control, 12 μL of PCR product without isolated plasmid DNA was added to the well. The ladder used containing 8 μL of nuclease-free water, 2 μL of loading dye, and 2 μL of GeneRuler™ 1kb molecular size DNA Ladder (Thermo Fisher Scientific Inc., MA, USA). The electrophoresis process runs at 80 V for 30 minutes using PowerPac™ Basic Power Supply (Bio-Rad Laboratories, Inc., USA). The gel was then visualized with Ultraviolet Trans illuminator (UVDI, Major Science Inc., USA). The positive result of bla_DHA-1 gene detection was shown by presence of 405 bp b...

Table of contents

• Cover Page
• Advances in Biomolecular Medicine
• Copyright Page
• Table of Contents
• Preface
• Iron overload intolerance in Balb/c mice
• Mapping of health care facilities in the universal coverage era at Bandung District, Indonesia
• Influence of phosphatidylcholine on the activity of SHMT in hypercholesterolemic rats
• Molecular detection of DHA-1 AmpC beta-lactamase gene in Enterobacteriaceae clinical isolates in Indonesia
• Correlation between natrium iodide symporter and c-fos expressions in breast cancer cell lines
• Fat mass profile in early adolescence: Influence of nutritional parameters and rs9939609 FTO polymorphism
• A study of the palatal rugae pattern as a bioindicator for forensic identification among Sundanese and Malaysian Tamils
• Effect of acrylamide in steeping robusta coffee (Coffea canephora var. robusta ) on memory function and histopathological changes of brain cells in male rats (Rattus norvegicus )
• Effects of aerobic exercise and a high-carbohydrate diet on RBP4 expression in rat skeletal muscle
• Correlation of physical activity and energy balance with physical fitness among the professors of the University of Padjadjaran
• Molecular biology of irreversible pulpitis: A case report
• Effect of different doses of X-ray irradiation on survival of human esophageal cells
• The effect of mesenchymal stem cells on the endothelial cells of diabetic mice
• Effects of selenium on SePP and Apo B-100 Gene expressions in human primary hepatocytes
• Inhibition of cAMP synthesis abolishes the impact of curcumin administration in the skeletal muscle of rodents
• Effect of Monosodium Glutamate (MSG) on spatial memory in rats (Rattus norvegicus )
• Mycobacterium tuberculosis load and rifampicin concentration as risk factors of sputum conversion failure
• Interleukin-22 serum in comedonal acne vulgaris: Proof of inflammation
• Pharmacokinetic optimization of the treatment of TB meningitis with TB drugs
• Iron-chelating effect of Caesalpinia sappan extract under conditions of iron overload
• The role of S.aureus and L.plantarum as an immunomodulator of IFNα macrophages and fibronectin dermal fibroblast secretion
• Exon globin mutation of β-thalassemia in Indonesian ethnic groups: A bioinformatics approach
• Serum immunoglobulin-E level correlates with the severity of atopic dermatitis
• Fatty liver in fasted FABP4/5 null mice is not followed by liver function deterioration
• The potential of seluang fish (Rasbora spp.) to prevent stunting: The effect on the bone growth of Rattus norvegicus
• Effect of cryoprotectants on sperm vitrification
• Mucoprotective effect of Trigona propolis against hemorrhagic lesions induced by ethanol 99.5% in the rat’s stomach
• Author index