Van Thi Thu Ha^1,2, Hieu Duc Nguyen¹, Minh Ngoc Nghiem¹, Son Thai Nguyen², Tram Bao Nguyen¹, Duc Minh Nguyen¹, Cuong Xuan Hoang³, Thuy Thi Bich Vo^1*

¹Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi, Vietnam
²Military Hospital 103, Vietnam Military Medical University, Hanoi, Vietnam
³Vietnam Military Medical University, Hanoi, Vietnam

*Corresponding Author: Thuy Thi Bich Vo, Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi, Vietnam; Email: [email protected]

Published Date: 28-02-2022

Copyright^© 2022 by Vo TTB, et al. All rights reserved. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Background: The over using of antibiotics in hospitals, communities and agriculture has raised drug resistance in Vietnam.

Objective: The study was to identify the multi antimicrobial resistance gene expression associated with extensively drug resistance phenotype in Klebsiella pneumonia (K.pneumoniae) isolated from clinical samples in the Military Hospital 103 in Vietnam.

Material and Method: Seventeen extensively drug resistant (XDR) strains were identified and measured antibiotic susceptibility by using Vitek 2 Compact System. Genes related to the antimicrobial resistant were screened using in-house multiplex PCR at Vietnam Institute of Genome Research.

Results: K.pneumoniae showed resistance to almost fifteen antibiotics with very high rates of over 70%, only sensitive to colistin 100%. There is a correlation between carpabenem resistance and XDR status in the K.pnemoniae group. Most of the K.pneumoniae isolates harbored the widely distributed ESBL gene groups. All the isolates carried more than 3 antibiotic resistance genes, 10 of which even carried 7 to 10 of the total 13 tested antibiotic resistance genes, equivalent to more than 13 antibiotic resistant phenotypes of all types.

Conclusion: Our study adds the significant of antibiotic resistance gene expressions that are showing the genotype and phenotype relations of XDR K.pneumoniae isolates. Future research should focus on the impact of these gene sources on the multidrug resistant phenotype of the patients and the genotypes can be used to predict the antibiotic sensitive phenotype of XDR K.pneumoniae isolates in hospital.

Keywords

Klebsiella pneumonia; Antibiotic Resistance Genes; Genotypic; Phenotype Resistances

Abbreviations

K.pneumoniae: Klebsiella pneumonia; XDR: Extensively Drug Resistant; MDR: Multiple Drug Resistance; PDR: Pan Drug Resistant; ESBL: Extended- Spectrum Beta-Lactamases; KPC: K.Pneumonia Pneumonia Carbapenemase; NDM: New Delhi Metallo-B-Lactamase; OXA: Oxacillinase; BMD: Broth Microdilution; MIC: Minimum Inhibitory Concentration; CLSI: Clinical And Laboratory Standard Institute; ICU: Intensive Care Unit; RT-PCR: Reverse Transcription Polymerase Chain Reaction; cDNA: Complementary Deoxyribonucleic Acid

Introduction

In the near decades, the increasing clinical incidence of antibiotic resistant bacteria is a serious issue for global health care [1]. The rapid dissemination of Multiple Drug Resistance (MDR) and Extensively Drug-Resistant (XDR) or Pan Drug Resistant (PDR) K.pneumoniae has recently recognized as an urgent public health threat, thus requiring immediate and aggressive actions [2]. Some clinical K.pneumoniae isolates can acquire of antibiotic resistance genes and intrinsic resistance of bacteria that has consequently limited treatment options for infections [3]. Currently, K.pneumoniae strains producing Extended-Spectrum Beta-Lactamases (ESBLs) and carbapenemases have spread globally [4]. The frequent outbreak of nosocomial infections due to these strains that is attributed to multiple drug resistance mechanisms [5]. The mechanisms involved when β-lactamase hydrolyzes β-lactam antibiotics or inactivates β-lactams have been investigated [6]. However, no precise molecular biological mechanisms underlying the antimicrobial drug resistance of K.pneumoniae have been reported [7]. In the previous several studies indicated that antibiotic resistance genes originate in microorganisms and subsequently integrate into the genome of other pathogens through transduction and/or transformation to produce a multitude of different antibiotic resistance strains that are especially dangerous for the course of treatment for patients [8-10]. Antimicrobial resistance genes in K.pneumoniae are also associated with high rates of morbidity and mortality in clinical patients [11]. Therefore, understanding the mechanisms involved in the antibiotic resistance of K.pneumoniae are highly required to warrant the development of novel antibiotics or alternative treatment strategies to decrease mortality of patients infected with K.pneumoniae.

Globally, β-lactam antibiotics are the most widely used in the treatment of many infections. However, the formation of the ESBLs group has made the problem of antibiotic resistance in bacteria very worrisome [12]. There are three main types of ESBL such as TEM, SHV and CTX-M, which are considered to be the most common and influential. These types of ESBLs are constantly evolving; increasing in number and more complex in characteristic. Currently, more than 600 different ESBL enzyme variants have been discovered, corresponding to more than 600 different ESBL-encoding genotypes [12,13]. Carbapenem is the widest antimicrobial spectrum and stable activity to against bacteria that produce AmpC β-lactamase and ESBL, therefore, this antibiotic is considered last treatment options in the treatment of infections caused by multiple antibiotics resistant gram-negative bacteria [14]. However, the rapid dissemination of carbapenemase genes, namely, K.pneumonia Carbapenemase (KPC), New Delhi Metallo-b-lactamase (NDM) and Oxacillinase (OXA) type carbapenemases is the major contributing factor to the rapid global spread of carbapenem resistance [15]. Therefore, it is necessary to have measures to manage the rational use of antibiotics in both hospitals and communities to limit antibiotic resistance, especially multidrug resistance, which is increasing in the community.

Methodology

Sample Collection

The seventeen XDR K.pneumoniae isolates were randomly selected from over 1000 total clinical isolates between 2015 and 2017 in the Military Hospital 103. The identification of K.pneumoniae isolate was done by analyzing colony morphology in special culture media, microscopic examination, performing biochemical testing, and Vitek 2 system (bioMérieux, Marcy l’Étoile, France).

Resistant Antibiotic Phenotype Identification

Antimicrobial susceptibilities were performed for samples by the Vitek 2 system and the AST-GN card, E-test and Broth Microdilution (BMD) methods (bioMérieux, Marcy l’Étoile, France). Colistin susceptibility assay was performed according to recommendations of joint CLSI-EUCAST guidelines [16]. The Minimum Inhibitory Concentration (MIC) values of the antibiotics carbapenem was measured for all isolates using E-test (bioM’erieux) according to the manufacturer’s instructions. The results were used to classify the isolates as either susceptible, intermediate or resistant to the tested antibiotics, based on Clinical and Laboratory Standard Institute (CLSI) breakpoints [17].

DNA Extraction and Species Identification

The DNA was extracted using a GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer guideline. The quality and quantity of DNA were assessed before sequencing.  The 16S rRNA gene of seventeen isolates was sequenced in the ABI3500 system (Applied Biosystem). The isolate sequences in this study and 23 published reference sequences including twenty two Enterobacteriae strains and one out group strain were aligned using MUSCLE algorithms. The maximum likelihood tree with T92+G substitution model and bootstrap 1000 were built to determine the genetic relationship. All analysis processes were performed using MEGA6 software [18].

Quantitative Reverse Transcription-PCR

For molecular analysis of antibiotic resistance determinants of XDR K.pneumoniae, cDNA samples and primers of target genes (Table 1) were prepared for RT-PCR according to the protocol description. Briefly, total RNA was extracted from bacterial culture media using Trizol (Thermo Fisher Science, USA). The concentration and quality of each RNA samples were determined by measuring absorbance at 260 nm. Complementary DNA (cDNA) was synthezed from 1 µg of total RNA. The volume of 1 µl cDNA was used for PCR performed using standard conditions: denatured at 95°C for 30 seconds, annealed at 52-60^oC for 30 seconds, extended at 72^oC for 1 min, and performed using 30 cycles. The PCR product of 16S rRNA (a housekeeping gene) was used as a control for variations in mRNA concentrations in the RT-PCRs. A volume of 8 µl of PCR product was loaded on 2% agarose gel and stained with ethidium bromide. The gel photograph was scanned using Quantity One program (Gel Doc EQ; Bio-Rad,Hercules, CA).

Table 1: Primer sets used for screening target genes.

Results

Characteristics of Clinical Samples

K.pneumoniae isolates were isolated from 14 male (84.4%) and 03 female (17.6%) patients. Most of K.pneumoniae were isolated from patients aged from 23 to 92 years old (median age at 59.53). Besides, K.pneumoniae samples were mostly isolated from Intensive Care Unit (ICU) (71.4%) and non-ICU (9.6%) (Table 2).

The 16S rRNA-based subspecies analysis was conducted to determine the genetic relationships of the strains using in research. The Fig. 1 shows phylogenetic species of all K.pneumoniae isolates closely related in a group of K.pneumoniae strains ATCC_13883, 11296, 13884 (77 – 97 bootrap values), and  belong to the family Enterobacteriaceae. Thus, all strains of K.pneumoniae isolated at the hospital have highly genetically similarity level with K.pneumoniae ATCC strains.

Table 2: Sociodemographic characteristics associated with 17 K. pneumoniae bacterial isolates from patients who were admitted to the Military Hospital 103.

Antibiotic Sensitivity and MIC of Isolates Studied

The isolates were screened against with 18 antibiotics. The results showed that all K.pneumoniae isolates were still susceptible to colistin (100%); highly resistant to ampicillin, sulfamethoxazole/trimethoprim, and ertapenem (82.4 – 94.1%); and completely resistant (100%) to ampicillin, piperacillin/tazobactam, cefotaxime, certapenem, cefepime, ciprofloxacin, and norfloxacin. In particular, one isolate (KP-208H) was resistant to 17/18 tested antibiotics, and 2 to 6 isolates were resistant to 12 from 15 antibiotics (accounting for 88.89 %), and the lowest resistance was also 8/18 of the antibiotics tested (KP-007D isolate).

Besides, a study was designed to determine in-vitro efficacy of Modified Hodge Test (MHT) for detection of carbapenemase production in K.pneumoniae isolates [19]. Out of a total 17 isolates which were showing intermediate or susceptible zone, i.e. 6 mm – 23 mm for meronem, 14/17 (82.4%) were positive for carbapenemase production by MHT. In addition, the strains used in the study all had similar MICs with imipenem, meropenem and ertapenem and all were very high compared to the CLSI recommended standard, up to 32 µg / mL (Supplemental Table 1).

Antibiotic Resistance Gene Profile

From the analysis of RT-PCR results, genes responsible for conferring aminoglycoside resistance, carbapenem resistance, beta-lactam resistance, fluoroquinolone, and folate pathway antagonist were presented across all of K.pneumoniae isolates (Fig. 2, Table 3). Briefly, the highest occurrence were genes belonging to the group of combined resistance to fluoroquinolone and folate pathway antagonist (OqxB gene appeared in 16/17 isolates, accounting for 94.12%; OqxA, Sul-1, and Sul-2 genes were 74.47, 58.82 and 29.41%, respectively), followed by aminoglycoside and fluoroquinolone combination resistance (aac(6′)-Ib-cr gene, appearing in 15/17 isolates, approximately 88.24%), and then the group with combined carbapenem and beta-lactam resistance (the blaNDM gene appeared in 9/17 isolates, accounting for 52.94%, other blaKPC and blaOXA genes were 41.18 and 11.76%, respectively). The highest resistance genes in single groups such as beta-lactam, or fluoroquinolone, or aminoglycoside were blaCTX-M (70.59%), blaSHV and qnrB (both of them 58.82%), blaTEM (35.29%), and the lowest was rmtB gene (only approximately 5.88%).

Analysis of carbapenem resistance gene expression (Table 4) showed that all XDR K.pneumoniae isolates possess carbapenem resistance genes, which could appear independently or in combination of multiple genes in the same ESBL-producing bacteria. In particular, all five genes (blaNDM, blaOXA, blaCTX-M, blaTEM, and blaSHV) were found in KB-016D isolate; four genes occurred in 3 isolates (KB-017D, KB-553M, and KB-011D). There were 13 isolates containing from 1 to 3 carbapenem resistance genes, of which 3 isolates carried 3 genes (17.65 %), 8 isolates carried 2 genes (47.06 %), and only 1 isolate (KB-289M) carrying a gene blaCTX-M (5.88%). The results showed an association between the prevalence of antibiotic resistant gene expression and drug resistant phenotype in some isolates of K.pneumoniae. However, there was also the opposite, KB-007D carried 4 genes for carbapenem resistance but was 100% sensitive to the antibiotics of this group, therefore, these antibiotic resistance gene expressions did not correlate with the number of antibiotics to which a particular isolate has shown resistance. The cross-resistance to antibiotics is generally a combination of mechanisms, osmolarity, targets and enzymatis changes. Therefore, further studies are needed.

Table 3: Distribution of multiple resistance genes identified among the isolates.

Table 4: The multiple carbapenem resistance gene profile of K.pneumoniae isolates.

Discussion

In this study, most of the XDR K.pneumoniae isolates were collected from male patients. There may be a association in poor lifestyle choices, smoking and alcoholism between male and female [20]. In addition, most of K.pneumoniae in this study were isolated from patients aged 23 to 92 years old. The differences in age distribution of patients may be related to the response of the immune system, such as under 40 years have stronger immune systems, and more pressure to fight to K.pneumoniae [21]. Besides, an increasing age leads to increase the comorbid illness cause of a higher risk of K.pneumoniae infection [22]. These results also indicated that K.pneumoniae isolates were mainly isolated from ICU and respiratory specimens (namely is sputum samples). John and Dawson emphasized that K.pneumoniae typically colonizes human mucosal surfaces of the oropharynx [23]. For this reason, K.pneumoniae is considered to be the most common cause of hospital acquired pneumonia in the Vietnam [24].

Antimicrobial resistance bacteria are commonly associated with nosocomial infections cause of the overuse of antibiotics, and without monitoring or control [25]. In our study, most of K.pneumoniae was showed a fully resistant to ampicillin, piperacillin/tazobactam, cefotaxime, ceftazidime, cefepime, ciprofloxacin and norfloxacin, whereas the most sensitive with colistin, amikacin, nitrofurantoin, and fosfomycin. The finding was higher than other results, Cepas, et al., reported that 40% of K.pneumoniae strains were resisted to ciprofloxacin and amoxicillin-clavulanic acid [26]. Manjula, et al., showed that 90.2% of K.pneumonia isolates were MDR, and high resistance to penicillin, cephalosporin, fluoroquinolone, aminoglycoside, and sulfonamide [27]. These MDR microbes are causing a great challenge in controlling infections, thus it is very important to monitor and optimize antibiotic use through antibiotic management programs [28].

The analysis of antibiotic resistance gene expression and the relationship between genotypes and multi-drug resistance phenotype of the K.pneumoniae isolates were performed.  This will provide a clearer and more indepth look at about the XDR capacity of these strains. Here, K.pneumoniae isolates had 13 difference resistance genotypes separating in four antibiotic phenotypes. Among the resistance against carbapenems K.pneumoniae isolates, the results have shown a predominant of blaCTX-M gene, then blaSHV, blaNDM, blaKPC, blaTEM, and a low percentage of blaOXA gene. In the previous studies, blaSHV and blaTEM were the two most common ESBL-coding genes in ESBL-producing bacteria. However, in recent years the blaCTX-M gene groups have become dominant [12,13]. The distribution of ESBL genes in our study was similar to the results of some recent studies in Vietnam [29,30]. Thereby, it shows that the distribution trend of genes encoding ESBL production in Vietnam is similar in the world, especially the common and widespread distribution of blaCTX-M instead of blaTEM and blaSHV, from there can confirm the flexible, unpredictable and difficult to control variation of antibiotic resistance in bacteria. In addition, there were isolates harboring about from 3 to 5 resistance genes encoding ESBL, also carrying on 2 to 6 other antibiotic resistance genes, and all were phenotypically resistant to 13 and 14 antibiotics of the tested groups. The presence of many genes encoding ESBL and other antibiotic resistance genes on the same strain of bacteria has changed the antibiotic resistance phenotype in a more complicated direction, resistance to multiple antibiotics at the same time, and dangerous multidrug resistance [31]. According to some previous studies, because it is located on a plasmid, ESBL is easily transmitted from one species to another or within the same species. Besides, ESBL-carrying plasmids also carry other antibiotic resistance genes, creating co-resistance, so bacteria with ESBL are not only resistant to β-lactams but also resistant to many antibiotics of other groups such as quinolones, sulphonamides and aminoglycosides [13].

The study results also showed a similarity in genotypic and phenotypic antibiotic resistances. Specifically, 16 isolates of XDR K.pneumoniae had 100% resistant phenotype with CIP and NOR (fluoroquinolone antibiotic group) all haboured OpxB genotype. The blaCTX-M genotype indicating for CTX-resistant phenotype was presented in 12/17 isolates that were completely resistant to CTX (beta-lactam antibiotic group). The similar isolates with 100% phenotype resistance to CAZ (beta-lactam group) also appeared 2 genes indicating CAZ resistance, which were blaSHV and blaTEM. The level of resistance to AN (an antibiotic belonging to the aminoglycoside group) was low (23.5%), equivalent to rmtB gene also appeared in only one isolate. The formation of resistance phenotypes in microorganisms is due to genetic changes in chromosomes or due to the reception of plasmids carrying resistance genes. This diverse mechanism of genetic information transmission contributes to the rapid spread of antibiotic resistance genes within species and to other bacterial species [13]. This leads to great difficulties for the selection of specific antibiotics in clinical treatment, although generations of antibiotics are constantly being researched and updated. Obviously, the emergence of strains of XDR K.pneumoniae resistant in the hospital is a dilemma for clinicians.

Conclusion

Taken together, our study has added a systematic analysis of 13 antibiotic resistance genes expression (belong to Carbapenem, Beta-lactam, Fluoroquinolone, Folate pathway antagonist, and Aminoglycoside groups), the association between multiple genotypes and XDR phenotypes in 13 K.pneumoniae isolates, and other clinical changes in antibiotic resistance of XDR K.pneumonia isolates in hospital. From the research results, there may be new suggestions to improve drug resistance and increase the effectiveness of treatment.

Acknowledgement

The authors thank to Department of Medical Microbiology, Military Hospital 103 for collecting samples. Vietnam Academy of Science and Technology support this research under grant number NCVCC40.06/22-22.

Conflicts of Interest

The authors declare that have no competing interest and not any conflict of interest.

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Article Type

Research Article

Publication History

Received Date: 05-02-2022
Accepted Date: 21-02-2022
Published Date: 28-02-2022

Copyright© 2022 by Vo TTB, et al. All rights reserved. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Vo TTB, et al. Analysis of Antibiotic Resistance Genes of the Extensively Drug Resistant Clinical Klebsiella pneumonia Isolates in Vietnam. J Clin Immunol Microbiol. 2022;3(1):1-15.

Figure 1: Phylogenetic tree was built using Maximum-Likelihood algorithm, bootstrap 1000. “Vietnam”: mean the seventeen multidrug-resistance K.pneumoniae isolated in the Military Hospital.

Figure 2: List of antibiotic resistance gene expression.

Table 1: Primer sets used for screening target genes.

Table 2: Sociodemographic characteristics associated with 17 K. pneumoniae bacterial isolates from patients who were admitted to the Military Hospital 103.

Table 3: Distribution of multiple resistance genes identified among the isolates.

Table 4: The multiple carbapenem resistance gene profile of K.pneumoniae isolates.

Supplemental Table 1: Phenotypic antimicrobial resistance pattern of the 17 K. pneumoniae isolates.