Research Article

 

Colistin-resistance and carbapenemase-producing among carbapenem-resistance E. coli and K. pneumoniae

Resistencia a la colistina y producción de carbapenemasas entre E. coli y K. pneumoniae resistentes a carbapenem

 

Ngoc Tran Bich1 https://orcid.org/0000-0002-2255-6398
Phu Truong Thien2* https://orcid.org/0000-0003-0868-2811
Loc Nguyen Tran Mai1 https://orcid.org/0000-0003-3232-3587
Dang Tran Ngoc1 https://orcid.org/0000-0002-5287-0265
Thao Nguyen Thi Ngoc2 https://orcid.org/0009-0004-1295-401X
Phuong Nguyen Thi Nam2 https://orcid.org/0009-0002-5305-4726
Tuan Dang Anh2 https://orcid.org/0009-0008-1677-2650
Van Tran Thi Hue1 https://orcid.org/0009-0007-0677-2553
Dat Ngo Quoc1 https://orcid.org/0000-0003-1461-0216

 

1University of Medicine and Pharmacy at Ho Chi Minh City. Ho Chi Minh City, Vietnam.
2Cho Ray Hospital. Ho Chi Minh City, Vietnam.

*Author for correspondence. Email: truongthienphu78@yahoo.com

 

 

ABSTRACT

Introduction: Carbapenemase-producing Enterobacterales (CPE) is one of the causes of community health-threatening, most common in Escherichia coli and Klebsiella pneumoniae. CPE has appeared in many countries, especially developing ones, but it has not been recognized in routine antibiograms. Also, assessing colistin susceptibility is a significant obstacle for both diagnostic and epidemiological because of technical difficulties. There is rare data about CPE and colistin-resistant Enterobacterales.
Objectives: Determine the prevalence of producing carbapenemase and colistin-resistance among K. pneumoniae and E. coli resistant to carbapenems.
Methods: In a cross-sectional survey, 129 K. pneumoniae and 31 E. coli strains, which demonstrated carbapenem resistance, were collected from inpatients at Cho Ray Hospital over a three-month period. Carbapenemase production was detected using the modified carbapenem inactivation method and EDTA-modified carbapenem inactivation method, and colistin resistance was detected using the broth microdilution method according to CLSI M100S34.
Results: Of 160 participants' strains, 80% of carbapenemase-producing K. pneumoniae and E. coli were detected; among these, Metallo beta-lactamase-producing was taken 69,5%. Of 31 colistin-resistant, 30 were K. pneumoniae, and only 1 E. coli was detected that was resistant to colistin.
Conclusion: Among participants' strains, carbapenemase-producing prevalence was very high. There were signs of a widespread extensively drug-resistant K. pneumoniae. Most of E. coli was susceptible to colistin.

Keywords: Klebsiella pneumoniae; Escherichia coli; carbapenemase; colistin; polymyxin E; drug resistance, bacterial.

RESUMEN

Introducción: Enterobacterales productoras de carbapenemasa (CPE) son una amenaza a la salud comunitaria, más común en Escherichia coli y Klebsiella pneumoniae. CPE aparecen en muchos países, especialmente en vías de desarrollo, pero no se reconocen en los antibiogramas rutinarios. Evaluar la susceptibilidad a la colistina es difícil, tanto para diagnóstico como epidemiología, por las dificultades técnicas. Hay pocos datos sobre Enterobacterales resistentes a la colistina y productoras de carbapenemasa.
Objetivos: Determinar la prevalencia de la producción de carbapenemasas y resistencia a la colistina entre K. pneumoniae y E. coli resistentes a carbapenemos.
Métodos: Estudio transversal; se recolectaron 129 cepas de K. pneumoniae y 31 cepas de E. coli, resistentes a carbapenemos, de pacientes hospitalizados en el Hospital Cho Ray, durante 3 meses. La producción de carbapenemasa se detectó mediante el método modificado de inactivación de carbapenem y el método modificado de inactivación de carbapenem con EDTA; la resistencia a la colistina se detectó mediante el método de microdilución en caldo, según CLSI M100S34.
Resultados: De las 160 cepas se detectó el 80 % de K. pneumoniae y E. coli productoras de carbapenemasa; entre estas, se encontraron MBLs en el 69,5 %. De 31 resistentes a colistina, 30 eran K. pneumaniae y solo se detectó 1 E. coli resistente a la colistina.
Conclusión: Entre las cepas de los participantes, la prevalencia de producción de carbapenemasa fue muy alta. Hubo signos de una K. pneumoniae extensamente resistente a los medicamentos. La mayoría de E. coli fue susceptible a la colistina.

Palabras clave: Klebsiella pneumoniae; Escherichia coli; carbapenemasa; colistina; polimixina E; resistencia a los medicamentos, bacteriana.

 

 

Received: 12/06/2024
Approved: 08/07/2024

 

 

INTRODUCTION

Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are 2 of the omnipresent Gram-negative bacilli belonging to the Enterobacterales order. They are common bacterium in the colon, but they could lead to many severe infection syndromes in humans, such as meningitis, gastrointestinal and urinary tract infections, septicemia, and others. In addition, most of them are multidrug-resistant.(1)

Although carbapenems are commonly used for treating severe infections, carbapenemase-producing Enterobacterales (CPE) is also one of the causes leading to community health-threatening.(2)Carbapenemase, a beta-lactamase, includes Ambler classes A, B, and D with serin beta-lactamase (SBL) and metallo beta-lactamase (MBL) encoded by both genes in chromosome and plasmid.(3) Along with the rapid spreading of CPE, the effectiveness of these last-resort drugs is decreasing.(4) Besides ceftazidime/avibactam, the first line of CPE treatment, the return of an old antibiotic, colistin, is salvation, and combined therapeutic regimens consisting of colistin are broadly used worldwide.(5)

In recent years, CPE has appeared in many countries, especially developing ones, but it has not been recognized in routine antibiograms.Also, assessing colistin susceptibility is a significant obstacle for both diagnostic and epidemiological because of technical difficulties.(5) The size of the colistin molecular structure is quite large, so colistin resistance cannot be detected by routine methods such as Kirby-Bauer or E-tests.(5)

Consequently, there is rare data about CPE and colistin resistance Enterobacterales, so research about multidrug resistance (MDR) should be conducted to support physicians in selecting the right antibiotic. Therefore, this study surveyed colistin-resistant and carbapenemase phenotypes in carbapenem-resistant E. coli and K. pneumoniae.

This study aims to determine the prevalence of producing carbapenemase and colistin-resistance among K. pneumoniae and E. coli resistant to carbapenems.

 

 

METHODS

Study design and participants

A cross-sectional study was conducted at Cho Ray Hospital in Ho Chi Minh City, Vietnam. This is a terminal hospital with diverse patients from the Southern provinces. 160 modulated isolates of K. pneumoniae and E. coli from October 2023 to December 2023, which show resistance to at least one antibiotic of carbapenems, were collected from inpatients in the clinical departments of Cho Ray Hospital.

Data collection

The samples were cultivated in Blood agar, MacConkey agar, chocolate agar, or chromogenic agar, depending on which type of specimens, and incubated from 16 to 18 hours at 35-37 °C to assess viability and purity. Then, using the Vitek MS System (BioMérieux, France) to identify bacteria, using the Vitek-2 compact system (BioMérieux, France) to detect carbapenem resistance bacteria strains. E. coli ATCC 25922 and K. pneumoniae BAA ATCC 1705 were used for quality control for bacterialidentification tests and chromogenic agar.

After bacterial strains were assessed in this study, each sample was coded with an ID, so the participants' information was secured to avoid bias errors in collecting data. Each isolate was stored in TSB 15% until used.

The modified carbapenem inactivation method and EDTA modified carbapenem inactivation method (mCIM/eCIM) were used to detect carbapenemase-producing Enterobacteriales. The test procedure and interpretation were performed according to CLSI M100 S34.(6) Bacteria would be considered to produce carbapenemase when they have positive results on the mCIM test. If these strains have positive results with eCIM, they would be detected as MBL, either, or SBL.

Colistin resistance was detected by the Broth microdilution method (BMD). The test procedure complied with the manufacturer's recommendation (Trek diagnostic), and the test interpretation followed the CLSI M100 S34.(6) Colistin is resistant to bacteria strains when MIC is > 2 µg/mL and colistin is intermediate to bacteria strains when MIC is ≤ 2 µg/mL.

Data analysis

The Student Version of IBM-SPSS version 20.0 was used to analyze data. Categorical variables about the characteristics of the study participantsare presented by frequency and percentages. The Chi-square test assessed associations between carbapenemase-producing type and colistin resistance. A p-value of less than 0.05 was considered statistically significant.

Ethical considerations

The study collected frozen bacterial strains isolated from patients who met the study's selection criteria and did not have any intervention on the patients. The University of Medicine and Pharmacy Ethics Committee at Ho Chi Minh City approved this research with the number contract 142/HĐĐĐ-ĐHYD.

 

 

RESULTS

Characteristics of the study participants

In the flow chart of colony participants, 162 samples were assessed for initial eligibility and selected to participate in this study. The figure (Fig. 1) was shown in the flow chart of participants throughout the study. Participant strains that were excluded (attached the reasons) were noted; there were 160 eligible participant strains.

 

 

In table 1, of the 160 colony participants from many specimens screened in the study, most came from elders (more than 60 years old), accounting for 53.7%. Among these, the prevalence of K. pneumoniae was more frequent than that of E. coli (80.6% and 19.4%), and most bacteria strains were resistant to a combo of ertapenem, imipenem, and meropenem, accounting for 93.8%.

 

 

Prevalence of carbapenemase

 

 

In table 2, 128 of 160 participant strains were identified as having carbapenemase with mCIM-positive results, accounting for 80% of the total sample. Specifically, of these 128 samples, there are 89 samples, accounting for 69.5% belonging to MBLs with eCIM-positive results, and others were SBLs.

Of 129 K. pneumoniae, 102 samples were identified as carbapenemase, accounting for 79.1%. Among these, 70 samples (68.6%) with MBLs were detected; the rest were SBLs.

Of 31 E. coli, 26 samples were identified as carbapenemase, accounting for 83.9 %. Among these, 19 samples (73.1%) with MBLs were detected; the rest were SBLs .

Prevalence of colistin resistance

 

 

In table 3, 31 of 160 participant strains were identified as colistin-resistant, accounting for 19.4% of the total sample. Of 129 K. pneumoniae, 30 samples were identified as carbapenemase, accounting for 23.3%. Among 31 E. coli, only 1 sample was identified as colistin resistance, accounting for 3.2%. There were statistical differences in colistin resistance between K. pneumoniae and E. coli with p= 0.011.

Correlation between carbapenemase and colistin-resistance

Of the 31 participant strains with colistin-resistant results, 14 were MBLs, 8 were SBLs, and others did not have any carbapenemase; there was no statistical difference between carbapenemase types and either having or not carbapenemase (table 4).

Among 30 colistin-resistant K. pneumoniae,14 were MBLs, and 7 were SBLs. Only 1 E. coli was resistant to colistin, which was detected as having SBLs.

 

 

DISCUSSION

The prevalence of CPE has been spreading rapidly and poses a problem with public health challenges and the healthcare system. As a developing country, Vietnam has faced the increasing prevalence of MDR bacteria, which could threaten to undermine the effectiveness of the last resort antibiotic.

This study collected samples from various specimens with 80% carbapenemase prevalence detected in K. pneumoniae and E. coli. These results aligned with Sharma K et al.(7) in India, in which this prevalence showed 86.1%.(7) In Egypt and China, Khattab S et al.(8) and Tsai YM et al.(9) study has a slightly lower 70.7% and 57.1%, respectively. The causes of these differences may stem from variations in ethnicity, living conditions, and antibiotic usage habits.

Specifically, among 128 CPE, 89 (69.5%) isolates producing MBLs were detected by using the mCIM/eCIM method. This result is reasonable, considering the epidemiology of the distribution of carbapenemase types in South and Southeast Asia.(10)

Metallo-carbapenemase, known as harboring NDM, VIM, or IMP enzymes, often shows resistance to a wide range of beta-lactams, including carbapenems. Newer antibiotics and beta-lactamase inhibitors, such as ceftazidime-avibactam, are ineffective against MBLs. However, aztreonam, which MBLs do not hydrolyze in combination with avibactam, can be considered a treatment option.(11) Besidesthat, combination therapy can enhance treatment efficacy and help prevent the development of further resistance. In developing countries like Vietnam, combining classic antibiotics such as colistin may be effective against some metallo-carbapenemase bacteria.(11) Nevertheless, colistin-resistant bacteria are present worldwide.

Bacterial strains that are resistant to both carbapenems and colistin are called extensively drug-resistant (XDR).(12) In this study, overall colistin resistance among K. pneumoniae and E. coli is 19.4%. Current result is similar to the study of Raunak Biret al. (15%).(13) Specifically, the number of colistin-resistant K. pneumoniae (CRKP) was significantly higher than E. coli (p= 0.011). Among 129 CRKP in current study, 30 strains (23,3%) resisted colistin. It was correlated with Bui Thanh Thuyet et al.(14) So, along with previous studies, this research was evidence of the rising rate of XDR in Vietnam's hospitals. There was only one colistin-resistant E.coli among participant strains, accounting for 3.3%. Therefore, initial antibiotic treatment with colistin could be considered for carbapenem-resistant E. coli.

Of 30 CRKP, there are 14 strains producing MBLs (45.2%) and 8 strains producing SBLs (25.8%). There was no statistical difference in colistin resistance between carbapenemase type and either having or not having carbapenemase. However, in the study of Elaskary SA et al.,(15) among colistin-resistant strains, there was a significant difference in carbapenemase gene prevalence, with SBLs being more predominant than MBLs in CRKP. Epidemiological differences across countries may account for this variation prevalence.

There are also some limitations. This research was about carbapenemase phenotype and could not show the exact genes that code for MBLs or SBLs. Furthermore, the sample size should come from a multicenter to be more significant in detecting MDR, especially in E. coli.

The prevalence of carbapenemase-producing strains among participants' strains was very high. There were signs of a widespread, extensively drug-resistant K. pneumoniae. Most E. coli strains were susceptible to colistin.

Based on the evidence from this research at Cho Ray Hospital, aztreonam combined with colistin could be recommended for treating diseases caused by CRE, especially E. coli.(16)

 

 

Acknowledgments

We give special thanks to the University of Medicine and Pharmacy at Ho Chi Minh City for funding and Cho Ray Hospital for assisting us with the auto-machines and facilities for the research project. Furthermore, we are honored to aid samples from the Microbiology Department, Cho Ray Hospital, in implementing our study.

 

 

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Conflict of interest

The authors declare that there is no conflict of interest.

 

Financial information

This research was funded by the University of Medicine and Pharmacy at Ho Chi Minh City under contract number 212 /2023 HĐ-ĐHYD dated 09/22/2023

 

 

Authors' contributions

Conceptualization: Ngoc Tran Bich.
Data curation: Loc Nguyen Tran Mai, Thao Nguyen Thi Ngoc, Ngoc Tran Bich.
Formal analysis: Dang Tran Ngoc, Ngoc Tran Bich, Loc Nguyen Tran Mai.
Research: Loc Nguyen Tran Mai, Thao Nguyen Thi Ngoc, Ngoc Tran Bich, Phu Thien Truong
Methodology: Loc Nguyen Tran Mai, Thao Nguyen Thi Ngoc, Ngoc Tran Bich, Phương Nguyen Thi Nam, Anh Dang Tuan.
Supervision: Phu Thien Truong, Dat Ngo Quoc.
Validation: Phu Thien Truong, Van Tran Thi Hue.
Writing - original draft: Ngoc Tran Bich, Phu Thien Truong.
Writing - review and editing: Ngoc Tran Bich.

 

 

Data availability

This research data is confidential according to the applicable confidentiality agreements and regulations and, therefore, cannot be publicly displayed or shared. The data are securely stored at the Integrated Planning Department at Cho Ray Hospital. Access to these data requires proper authorization. If you have any questions or need further information, please contact Ngoc Tran Bich at bichngoctran@ump.edu.vn.

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