The Molecular detection of bla_VIM in carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex infections in Indonesia

Heriyannis Homenta¹*, Fredine Esther Silvana Rares¹, Olivia Amelia Waworuntu¹,

Lewis Fidel Matandung², Sulaiman Faiz Sandjaya², Bellanty Costanty Togas²,

Julyadharma Julyadharma³, Hani Susianti⁴, Dewi Santosaningsih⁵,

Noorhamdani Noorhamdani⁵

¹Department of Clinical Microbiology, Faculty of Medicine, Sam Ratulangi University, Manado, Indonesia.

²Bachelor Program, Faculty of Medicine, Sam Ratulangi University, Manado, Indonesia.

³Laboratory of Clinical Microbiology, Prof. Dr. R. D. Kandou Hospital, Manado, Indonesia.

⁴Department of Clinical Pathology, Faculty of Medicine, Brawijaya University/Dr. Saiful Anwar Hospital, Malang, Indonesia.

⁵Department of Clinical Microbiology, Faculty of Medicine, Brawijaya University/Dr. Saiful Anwar Hospital, Malang, Indonesia.

*Corresponding Author E-mail: herihomenta@unsrat.ac.id

ABSTRACT:

Background: Bacteria of carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRABC) is a significant pathogen causes illnesses acquired via healthcare around the world, however little is known regarding the molecular resistant gene of A. baumannii-calcoaceticus complex bacteria resistant to carbapenem in Indonesian healthcare facilities. This study is aimed to identify the carbapenemase resistant gene of CRABC in two referral care hospitals in the islands of North Sulawesiand East Java, Indonesia. Methods: The Vitek2^® system was used to identify the CRABC pathogen that were obtained from standard medical cultures, which included blood, lower respiratory tract, wound and urine samples from patients treated in the medical wards, intensive care unit (ICU), surgical wards, neonatal intensive care unit (NICU), and cardiovascular intensive care unit (CVICU) in two referral care hospitals in Manado, North Sulawesi, and Malang, East Java. Using PCR, we identified the presence of the carbapenemase resistance genes bla_GES, bla_VIM, bla_IMP1, and bla_OXA-48 carbapenemase resistance genes. Results: Seventy threeA. baumannii-calcoaceticus complex carbapenem resistance bacteria were collected. The specimens from the lower respiratory tract specimens, the isolates of the carbapenem-resistant A. baumannii-calcoaceticus complex were frequently found.In this study, we detected the bla_VIMcarbapenemase resistance genes from blood and sputum samples in tertiary care hospital in Malang. Conclusions: We identified the bla_VIMcarbapenemase resistance genes were found in the referral care hospitals in Malang, East Java, Indonesia. Awareness of the national prevention and surveillance system is isolated by the prevention and reduction of CRABC transmission.

KEYWORDS: Acinetobacter baumannii-calcoaceticus complex, carbapenemase, carbapenem-resistant gene, bla_VIM, Indonesia.

INTRODUCTION:

Gram-negative bacteria, namely Acinetobacter baumannii (A. baumannii), recently recognized as an important pathogen causing healthcare-associated infections^1,2. Acinetobacter genus had three related species due to the phenotypic similarity that is A. baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii as A. baumannii-calcoaceticus complex³.

Bacteria ofA. baumannii-calcoaceticus complex produce carbapenemase⁴, resulting the emergence of carbapenem-resistant A. baumannii-calcoaceticus complex (CRABC) due to the frequent use of carbapenem antibiotic⁵. CRABC infections are a significant public health problem due to the limited antibiotic therapy available, resulting in higher morbidity, mortality, and cost⁶. Furthermore, some data suggest that infections caused by multiple drug resistant organism result in more terrible clinical results than those caused by vulnerable strains^7-⁹.

Previous studies reported that A. baumannii resistant to carbapenemisolates were frequently found among sputum (30.4-41.4%), followed by blood (17.3-23.9%), wound (7.6-25%), and urine (3.3-7.4%)^10-¹².CDC reported 700 death from 8500 cases nosocomial infections caused by A. baumannii bacteria¹³.

Data regarding the resistance genes of CRABC in Indonesia is scarce, likely influenced by a variety of factors, including climate, equipment, disease, environment, sterilization and treatment^14,¹⁵. This study aims to explore and determine the profiles of carbapenem resistant genes of CRABC in specific place and periods during study withinProf. dr. R. D. Kandou Hospital in Manado, dr. Saiful Anwar Hospital in Malang, Indonesia.

MATERIALS AND METHODS:

Setting and study design:

This study involved two referral hospitals that is Prof. Dr. R. D. Kandou Hospital in Manado from June to November 2019 and dr. Saiful Anwar Hospital in Malang from March to August 2019¹⁶. In the two tertiary care hospitals, clinical samples were gathered from patients admitted in themedical wards, intensive care unit (ICU), surgical wards, cardiovascular intensive care unit (CVICU), andneonatal intensive care unit (NICU).

Bacterial isolates:

Based on clinically indicated culture in hospitalized patients, isolates of the A. baumannii-calcoaceticus complex were obtained from wound, urine, blood,andlower respiratory tract. Each hospital in this study, the method of isolation is a standard diagnostic procedure that is anonymously analyzed. For clinical culture using the A. baumannii-calcoaceticus complex, only one clinical specimen per patient was collected for this study.

The VITEK2^® system by bioMérieux, Lyon, France was used for the identification and antibiotic sensitivity test¹⁷^,¹⁸. and to fulfill as CRABC used 10µg imipenem antibiotic disc subsequently analysed according to the guidelines of CLSI 2021^19,²⁰. The isolates of CRABC were stored at -80°C in trypticase soy broth containing 10% glycerin until it was further characterized.

DNA extraction:

The EcoSpin Bacterial Genomic DNA Kit was utilized to isolate bacterial DNA. The samples of DNAextraction were kept in a -20°C storage. The presence of seventy three isolates for carbapenem resistant genesof CRABC has been described elsewhere²¹.

Carbapenemase genes identification:

PCR was performed to detect the resistance genes for Ambler class A (bla_GES), Ambler class B (bla_VIM, and bla_IMP1), and Ambler class D (bla_OXA-48) as previously describedas shown in Table 1²².

RESULTS:

A total of 73 CRABC isolates were collected from inpatients for clinical cultures of in Manado and Malang, with 30 isolates from Manado and 43 isolates from Malang. Lower respiratory tract specimens contained the most CRABC isolates (42/73 [57.5%]), followed by pus (12/73 [16.4%]), blood (11/73 [15.1%]), and urine (8/73 [10.9%]).

Carbapenemase production tests on 73 samples from Prof. dr. R. D. Kandou hospital and dr. Saiful Anwar hospital lead to positive results for bla_VIM gene identification of CRABC isolates from blood specimen (sample of N7) and sputum specimen (sample of N9) in dr. Saiful Anwar Hospital, Malang, instead negative results for bla_GES, bla_IMP1 and bla_OXA-48as shown in Table 2 and Figure 1.

Table 2. Carbapenemase production of CRABC in two tertiary care hospitals Malang and Manado

Hospital	Clinical specimen	Total specimen	Carbapenemase Resistance Genes
Hospital	Clinical specimen	Total specimen	**bla_GES**	**bla_VIM**	**bla_IMP1**	**bla_OXA-48**
Dr. Saiful Anwar Hospital, Malang	Blood	9	0	1	0	0
	Urine	5	0	0	0	0
	Sputum	28	0	1	0	0
	Pus	1	0	0	0	0
Prof. Dr. R. D. Kandou Hospital, Manado	Blood	2	0	0	0	0
	Urine	3	0	0	0	0
	Sputum	14	0	0	0	0
	Pus	11	0	0	0	0

bla_GES: Guiana extended spectrum, bla_VIM: Verona integron-encoded metallo-b-lactamases, bla_IMP1: imipenemase1, bla_OXA-48: Oxacillinase-48. blaVIM gene identification of CRABC isolates from blood specimen (sample of N7) and sputum specimen (sample of N9) in dr. Saiful Anwar Hospital, Malang

Figure 1. bla_VIM gene identification of CRABC isolates from dr. Saiful Anwar Hospital, Malang.

Lane 1 = DNA ladder; lane 2 = negative control; lane 3 = positive control Of bla_GES; lane 4 = positive control Of bla_VIM; lane 5 = positive control Of bla_OXA-48; lane 6 = positive control Of bla_IMP1; bla_VIM was detected in lane 13 and lane 15; other isolates were negative for gene resistance.

DISCUSSION:

CRABC bacteria are known to cause nosocomial infections, and there has been a notable increase in such infections, accompanied by antibiotic resistance, especially carbapenem antibiotics, Over the last decade, it has emerged as a globally recognized multidrug-resistant bacteria, resulting in increased costs, longer treatment times, higher morbidity, and mortality^1,².

So far, we know that this present study was our the first study to identify four resistance genes (bla_GES, bla_VIM, bla_IMP1, bla_OXA-48) in CRABC. The present research was carried out in two referral care hospitals in Indonesia, that is Prof. dr. R. D. Kandou Hospital in Manado, North Sulawesi, and dr. Saiful Anwar Hospital in Malang, East Java.

In our present study, the results from N7 and N9 sample obtained from blood and sputum specimen are extensively drug resistant (XDR) sample from dr. Saiful Anwar Hospital, Malang.This differs from the results of previous studies, where the isolates were classified as multidrug-resistant (MDR) in Jakarta, Surabaya^17,¹⁸, Egypt^23,²⁴, Taiwan²⁵, Nepal²⁶,and Los Angeles²². These findings suggest an increasing rate of antibiotic resistance^27,²⁸ and a broadly of carbapenemase resistance genes from CRABC bacteria^29,³⁰, including in dr. Saiful Anwar Hospital, Malang. The N7 and N9 sample detected bla_OXA-23, positive mCIM and metallo-β-lactamase for the previos research²¹.The N7 and N9 sample were grouped into MT3, ST-642 and CC1, as indicated in prior research³¹.

In our present study, we identified the carbapenemase resistance gene bla_VIMin samples N7 and N9 obtained from blood and sputum at dr. Saiful Anwar Hospital, Malang. These results align with findings in Saudi Arabia³²and Surabaya, Indonesia³³where bla_VIMwas also detected. However, it differs from South Africa, where bla_VIM despite sharing the same ST1 clonality³⁴, as well as from Surabaya¹⁸, and Jakarta¹⁷.

In this present study, none of other genes bla_GES, bla_IMP1, and bla_OXA-48, were identified in CRABC isolates. However, carbapenem resistance can occur due to the presence of other resistance genes, and/or other resistance mechanisms such as efflux pumps³⁵ and biofilm formation³⁶. These variances may be related to human migration between nations or continents³⁷,and variations in climate, environment, equipment, sterilization, disease, and treatment options between locations have resulted in a diversity of resistant genes in A. baumannii bacteria^14,¹⁵.

Our study limitations for the current study. First, whereas it's being carried out at two referral care hospitals in Indonesia, It is still not considered national statistics. It could, however, be utilized as a point of reference to determine the CRABC molecular resistance gene in Indonesia. Second, because of restricted resources, we did not undertake resistance gene analysis on all clinical specimens from Prof. Dr. R. D. Kandou hospital, and dr. Saiful Anwar Hospital.

CONCLUSION:

In conclusion, we detected that bla_VIM was found in this study, however astatewide surveillance system is essential to improved awareness about the resistance gene of CRABC and to create a strategy to prevention, control and restrict the spread of CRABC in Indonesian health service facilities.

CONFLICTS OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENTS:

The authors would like to thank the funding of first author’s provided by Ministry of Education, Culture, Research and Technology Republic of Indonesia, especially The Institute for Research and Community Service, Sam Ratulangi University, research budget for 2023.

We are thankful to Board of Directors of Dr. Saiful Anwar Hospital, Malang, Indonesia; Board of Directors of Prof. Dr. R. D. Kandou Hospital, Manado, Indonesia; Laboratory of Microbiology in Dr. Saiful Anwar Hospital, Malang, Indonesia; Laboratory of Microbiology in Prof. Dr. R. D. Kandou Hospital, Manado, Indonesia; Department of Clinical Microbiology, Faculty of Medicine, University of Sam Ratulangi, Manado, Indonesia; Department of Clinical Microbiology, Faculty of Medicine, Brawijaya University, Malang, Indonesia; The Institute of Tropical Disease, Airlangga University, Surabaya, Indonesia.

ETHICS APPROVAL:

The ethical committee approved this study in health research of dr. Saiful Anwar Hospital, Malang (No. 400/059/K.3/302/2019), and Prof. Dr. R. D. Kandou Hospital, Manado (No. 054/EC-KEPK/IV/2019).

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Received on 22.11.2023 Modified on 12.03.2024

Research J. Pharm. and Tech 2024; 17(10):5031-5035.

DOI: 10.52711/0974-360X.2024.00773