Dissemination of Carbapenem-Resistance among Multidrug Resistant Pseudomonas aeruginosa carrying Metallo-Beta-Lactamase Genes, including the Novel blaIMP-65 Gene in Thailand

Piyatip Khuntayaporn; Wariya Yamprayoonswat; Montri Yasawong; Mullika Traidej Chomnawang

doi:10.3947/ic.2019.51.2.107

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Khuntayaporn, Yamprayoonswat, Yasawong, and Chomnawang: Dissemination of Carbapenem-Resistance among Multidrug Resistant Pseudomonas aeruginosa carrying Metallo-Beta-Lactamase Genes, including the Novel blaIMP-65 Gene in Thailand

Original Article

Infection & Chemotherapy 2019; 51(2): 107-118.

Published online: 24 May 2019

DOI: https://doi.org/10.3947/ic.2019.51.2.107

Dissemination of Carbapenem-Resistance among Multidrug Resistant Pseudomonas aeruginosa carrying Metallo-Beta-Lactamase Genes, including the Novel bla_IMP_-₆₅ Gene in Thailand

Piyatip Khuntayaporn¹

, Wariya Yamprayoonswat^1,²

, Montri Yasawong²

, Mullika Traidej Chomnawang¹

¹Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.

²Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok, Thailand.

Corresponding Author: Piyatip Khuntayaporn, PhD. Department of Microbiology, Faculty of Pharmacy, Mahidol University, 447 Sri Ayudthaya Rd; Rachthevi, Bangkok 10400, Thailand. Tel: +66-2-644-8692, Fax: +66-2-644-8692, piyatip.khn@mahidol.edu

Received 14 July 2018 Accepted 27 December 2018

The Korean Society of Infectious Diseases and Korean Society for Antimicrobial Therapy

(open-access, https://creativecommons.org/licenses/by-nc/4.0/):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Pseudomonas aeruginosa is considered as one of the most emerging threats in this century. Serious infections caused by this pathogen are often treated by carbapenems which are the last resource of antibiotics. Metallo-beta-lactamases (MBLs) production is one of the most important carbepenem resistance mechanisms and is usually related with nosocomial infections caused by P. aeruginosa. This study was aimed to determine the prevalence of MBL genes and distribution pattern of MBLs producing P. aeruginosa strains in Thailand.

Materials and Methods

Specific primers were designed to detect MBL genes including IMP-, VIM-, and NDM-type MBL genes. Multilocus sequence typing method was used to determine the dissemination pattern of carbapenem-resistance among multidrug-resistant (CR-MDR) P. aeruginosa.

Results

A total of 153 P. aeruginosa clinical isolates were characterized as CR-MDR. Among those, 31 P. aeruginosa clinical isolates (20.3%) presented metallo-beta-lactamase genes which could be divided into VIM-type (14 strains) and IMP-type (17 strains). bla_IMP_-₁, bla_IMP_-₁₃, bla_IMP_-_14a, and bla_VIM_-₂ genes were detected. Moreover, a novel IMP-type MBL, bla_IMP_-₆₅ was discovered and it was demonstrated to be the unique group of MBLs in Thailand. It was of interest that ST235 was the major ST type in Thailand followed by ST964 and ST111 and ST235 was detected in both MBL harboring and non-MBL harboring strains.

Conclusion

This study reported the dissemination of MBL gene including novel MBL, bla_IMP-65. This study was also demonstrated major ST of P. aeruginosa which was ST235, followed by ST964 and ST111. Moreover, it is also the first report on many P. aeruginosa STs in Thailand: ST273, ST292, ST621, ST1584, and ST1816 which emphasized the dissemination trait difference of MBLs harboring P. aeruginosa in Thailand.

Keywords: Pseudomonas aeruginosa, Beta-lactamases, Multilocus sequence typing, Antibiotic resistance, ST235

Introduction

Increasing of antibiotic resistance situations has raised an awareness around the world. A group of bacteria which had been recognized as threats of this century was named Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species [1]. Recently, WHO announced 12 bacteria that posed the greatest threat to human health. Among those, carbapenem-resistant Pseudomonas aeruginosa, Extended-spectrum β-lactamase (ESBL)-producing carbapenem-resistant Enterobacteriaceae, and carbapenem-resistant Acinetobacter baumannii were critical priority [2]. P. aeruginosa also belongs to this group because its resistance mechanisms such as efflux pumps, loss of porins, and production of beta-lactamase enzymes [3]. The overexpression of MBLs is also one of resistance mechanisms frequently found in carbapenem-resistant P. aeruginosa especially in severe infection.

To discriminate differences between each bacterial strains, multilocus sequence typing (MLST) is now recognized as a common tool using seven housekeeping genes [4]. This method was firstly introduced in 1998 and demonstrated major advantages in both macro- and micro-epidemiology with moderate to high discrimination power over many methods [4]. MLST method has been used in many pathogenic bacteria including P. aeruginosa which was introduced in 2004 [5]. Sequence type (ST) 235, ST111 and ST175 were considered as the majority of P. aeruginosa clinical isolates [6]. In Asia, there were some studies reported MLST of MBL-producing P. aeruginosa, mainly from China and Japan [7 8 9 10]. However, some studies showed the difference in ST prevalence, such as ST244 in China [7 8 9]. ST357 also demonstrated high prevalence as the second most ST [10 11]. Even though MBLs producing P. aeruginosa have been isolated in Thailand, only one study identified P. aeruginosa isolates of ST235 harboring bla_VIM_-₂ [12]. Therefore, the objective of this study was to investigate the dissemination traits of P. aeruginosa harboring MBLs including novel types of MBLs, bla_IMP_-₆₅, collected from hospitals across Thailand.

Materials and Methods

1. Bacterial culture collection

Carbapenems-resistant P. aeruginosa clinical isolates were collected from patients in eight hospitals across five regions of Thailand with human ethical approval from Mahidol University Institutional Review Board (Certificate No. MU-IRB 2011/025-0102). All hospitals are tertiary or university hospitals. A total of 153 P. aeruginosa clinical isolates were characterized as carbapenems resistance among multidrug resistance. Multidrug resistance (MDR) criteria in this study was defined as non-susceptible to at least 3 of 5 drug groups which used in Pseudomonas infection treatment including including anti-pseudomonal penicillin (piperacillin), cephalosporin (ceftazidime), carbapenems (imipenem and meropenem), fluoroquinolone (ciprofloxacin), and aminoglycoside (gentamicin). Carbapenems resistance (CR) was defined by being non-susceptible to at least one carbapenem [13 14]. The susceptibility of P. aeruginosa clinical isolates was confirmed in the research laboratory by the disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.

2. Phenotypic screening for metallo beta-lactamase

Phenotypic screening for metallo-beta-lactamase (MBL) enzyme was performed by diffusion method divided into 2 major methods which were double-disk synergy test (DDST) and combine disk test (CD) using EDTA as metallo beta-lactamase inhibitor [15].

3. Genotypic detection of metallo beta-lactamase gene and detection of integronI gene

Metallo beta-lactamase genes were divided into 3 groups for IMP-type MBLs and 2 groups for VIM-type MBLs depended on genotypic relationship of metallo beta-lactamase genes [16]. NDM-type MBLs were also included in this study. Each set of primers were designed to detect MBL genes in each group (Table 1). bla_IMP_-₁, bla_VIM_-₂ and bla_NDM_-₁ were used as controls. Whole-cell lysates obtained from clinical isolates were used as DNA templates for IMP-, VIM- and NDM-type MBLs detection by PCR method. All IMP-type PCR products were further identified for specific types of IMP-gene by PCR with IMP-G, IMP-H and IMP-13 primers. All PCR products were submitted for sequencing. Nucleotide sequences were compared with sequence databases using BLAST (http://blast.ncbi.nlm.nih.gov/).

Table 1

List of primers designed in this study

Primer	Nucleotide sequence (5′–3′)	Detectable gene	Group
IMP-AF	GTT AAC GGG TGG GGC GTT G	IMP-1, IMP-3, IMP-4, IMP-5, IMP-6, IMP-7, IMP-9, IMP-10, IMP-15, IMP-25, IMP-26	IMP-A
IMP-AR	AGC CAC TCT ATT CCG CCC GT		IMP-A
IMP-BF	TTG AAG AAG TTA ACG GGT GGG G	IMP-1, IMP-11, IMP-16, IMP-21, IMP-22	IMP-B
IMP-BR	TTT CAA GAG CGA CGC ATC TCC	IMP-1, IMP-11, IMP-16, IMP-21, IMP-22	IMP-B
IMP-CF	GGT TTG TGG AGC GCG GCT A	IMP-1, IMP-2, IMP-8, IMP-12, IMP-13, IMP-14, IMP-18, IMP-19, IMP-20, IMP-24	IMP-C
IMP-CR	CCT TTA ACA GCC TGT TCC CAT GT		IMP-C
VIM-DF	TAG CGG TGA GTA TCC GAC AGT	VIM-2, VIM-3, VIM-6, VIM-8, VIM-9, VIM-10, VIM-11, VIM-14, VIM-15, VIM-16, VIM-17, VIM-18, VIM-20, VIM-23, VIM-24	VIM-D
VIM-DR	TGC TTC CGG GTA GTG TTG TTG		VIM-D
IMP-GF	GCT ACC GCA GCA GAG TCT TT	IMP-1, IMP-10, IMP-30, IMP-52	IMP-G
IMP-GR	CAG GCA ACC AAA CCA CTA CG	IMP-1, IMP-10, IMP-30, IMP-52	IMP-G
IMP-HF	GGT TGG AGT GTG GTC ACT AAA C	IMP-14, IMP-14a, IMP-32, IMP-48, IMP-65	IMP-H
IMP-HR	CCA AAT AGC CAA GAC CGT CC	IMP-14, IMP-14a, IMP-32, IMP-48, IMP-65	IMP-H
IMP-13F	AAC ACG GTT TAG TGG TGC TTG T	IMP-13
IMP-13R	TTA GTT ACT TGG TGA TGA TGT TT	IMP-13
IMP-NF	ATG AAA AAA TTA TTT GTT TTA TGT	IMP-14, IMP-14a, IMP-32, IMP-48, IMP-65
IMP-NR	TTA GTC GCT TGG CTG TGA	IMP-14, IMP-14a, IMP-32, IMP-48, IMP-65
NDM-F	CAG CGC AGC TTG TCG	NDM-1, NDM-2, NDM-3, NDM-5, NDM-7
NDM-R	TCG CGA AGC TGA GCA	NDM-1, NDM-2, NDM-3, NDM-5, NDM-7
mutL-F	AGG TTC GCG ACC TGT TCT T	mutL
mutL-R	GGA CTC TCC AGC ACG CTC T	mutL
nuoD-F	CAC CGA GTT CGA TCC CTA CT	nuoD
nuoD-R	GGC ATC CAG TCG AGG AAC T	nuoD

IMP-AF, IMP-AR, IMP-BF, IMP-BR, IMP-CF, IMP-CR, VIM-DF, VIM-DR, IMP-GF, IMP-GR, IMP-HF, IMP-HR, IMP-HR, IMP-13F, IMP-13R, IMP-NF, IMP-NR, NDM-F, NDM-R, mutL-F, mutL-R, nuoD-F, and nuoD-R are primer name assigned for each group of detected genes.

IMP, imipenemase; VIM, verona integron-encoded metallo-β-lactamase; NDM, New Delhi metallo-beta-lactamase.

All CR-MDR P. aeruginosa were submitted for integronI detection by PCR method. The specific primers, Int1-F and Int1-R, of integronI gene were used as previously described [17].

4. Amplification of new allele of IMP- metallo beta-lactamases

IMP-N primers were designed for a full gene amplification. bla_IMP_-₁₄ was used as template because new allele of MBLs present high similarity sequences to bla_IMP_-₁₄. The new allele of metallo-beta-lactamase sequence was translated to protein sequence by BioEdit program. A full sequence of new metallo-meta-lactamase allele was submitted to assign as a novel allele at NCBI database. DNA Sequences of novel allele were analyzed and compared with similar sequences which were bla_IMP_-₁₄, bla_IMP_-_14a, bla_IMP_-₃₂ and bla_IMP_-₄₈.

5. Phylogenetic analysis

Nucleotide sequences of the novel bla_IMP_-₆₅ gene (KY315991) and other IMP type genes were aligned based on iterative refinement method using MUSCLE version 3.8.31 [18]. Phylogenetic tree of IMP type genes was reconstructed based on the randomized accelerated maximum likelihood. General time reversible model (GTR) with gamma distribution (G) and invariant sites (I) was a selected DNA substitution model for the phylogenetic analysis using RAxML version 8 [19]. The confidence values on phylogenetic tree were presented as percentage of bootstrap values (1,000 bootstrap replicates).

6. Multilocus sequence typing for metallo beta-lactamase producing P. aeruginosa

MBL harboring P. aeruginosa were selected as representatives when clinical isolates harbored the same MBL gene and demonstrated the same PFGE pattern [14]. A total of 14 clinical isolates P. aeruginosa was chosen and characterized for sequence typing (ST) by MLST method. Seven meropenem non-susceptible among MDR P. aeruginosa clinical isolates were chosen for comparison.

P. aeruginosa clinical isolates were characterized for molecular typing. MLST was performed as described previously with some modifications [5]. Briefly, seven housekeeping genes (ascA, aroE, guaA, mutL, nuoD, ppsA, and trpE), were amplified and sequenced. The nucleotide sequences were determined and compared with MLST database to assign the allele of these genes and STs (http://pubmlst.org/paeruginosa/). Primers for mutL and nuoD genes were modified in this study as showed in Table 1.

Results

1. Detection of integronI and metallo beta-lactamase genes

All CR-MDR P. aeruginosa were identified for phenotypic resistance pattern by diffusion method and genotypically detected for intI gene by PCR. One hundred and thirty six (88.9%) clinical isolates of P. aeruginosa were positive for int1 gene. Phenotypic screenings with EDTA were used to detect the presence of metallo beta-lactamases. One hundred and four clinical isolates (68.0%) of CR-MDR P. aeruginosa were positive by CDT and/or DDST. These strains were further subjected for metallo beta-lactamase genes detection by PCR with specific primers. Total of 31 (20.3%) P. aeruginosa clinical isolates were found to present metallo beta-lactamase genes which could be divided into VIM-type for 14 strains and IMP-type for 17 strains. For VIM-type, all 14 strains were detected by VIM-D primers and PCR products were sequenced and identified as bla_VIM_-₂. However, NDM-type MBL could not be detected in this study.

For IMP-type MBLs, all 17 strains were detected by IMP-C primers. Those strains were amplified with IMP-G, IMP-H and IMP-13 primers to identify specific types of IMP-MBLs. PCR products were sequenced and found 8 bla_IMP_-₁, 6 bla_IMP_-₁₄ and only one bla_IMP_-₁₃ harboring strains. However, two strains were carried unidentified MBLs.

2. Novel allele of IMP-type MBLs

Those two strains carried unidentified IMP-type MBLs were further investigated. IMP-14 primers were designed based on bla_IMP_-₁₄ sequence because unidentified MBLs could present high similarity sequences to bla_IMP_-₁₄. The novel gene was mutated at position 279 and 301 from bla_IMP_-₁₄ gene. Therefore, translated protein was changed from threonine (T) to alanine (A) at position 101 (Fig. 1). The novel gene was deposited and assigned as bla_IMP_-₆₅ by the NCBI (accession number KY315991). Dissemination of MBL genes detected in P. aeruginosa across Thailand was showed in Fig. 2. Translated protein sequences of IMP14, IMP-14a, IMP-32, IMP-48 and IMP-65 were compared as showed in Fig. 1. These sequences presented high similarity with one or two amino acids change.

Figure 1

Translated protein sequences of IMP-14, IMP-14a, IMP-32, IMP-48 and IMP-65. The difference sequences were indicated in different colors.

IMP, imipenemase.

Figure 2

Dissemination of MBL genes detected in P. aeruginosa across Thailand. The type of MBL genes were followed by number of samples.

MBL, metallo-β-lactamase; IMP, imipenemase; VIM, verona integron-encoded metallo-β-lactamase.

3. Phylogenetic Analysis

The phylogenetic tree represented the relationship among the novel IMP-type gene (bla_IMP_-₆₅) and other IMP-type genes (Fig. 3). There were three groups of the IMP-type genes (IMP-type group I, II and III) presented on the tree. The novel IMP-type gene was affiliated with the IMP-type group I. bla_IMP_-₃₂, bla_IMP_-₄₈, bla_IMP_-_14a, bla_IMP_-₁₄ and bla_IMP_-₅₄ were the closest relative genes of bla_IMP_-₆₅. This IMP-type subgroup had been supported by 100% of bootstrap (Fig. 3). It was clearly demonstrated that Thailand was the origin of bla_IMP_-₁₄, bla_IMP_-_14a, bla_IMP_-₄₈ and bla_IMP_-₆₅ isolates (Supplementary Table 1).

Figure 3

Evolutionary relationships of IMP-type genes. The phylogenetic tree reconstructed using the randomized accelerated maximum likelihood method with the GTR+I+G model of DNA substitution. The values associated with nodes correspond to the bootstrapping support in %. Bootstrap value lower than 50% was not represented on the clades.

IMP, imipenemase; GTR, general time reversible model; I, invariant sites; G, gamma distribution; DNA, deoxyribonucleic acid.

4. MLST profile analysis

Based on MLST analysis, a total of 11 clinical isolates of MDR P. aeruginosa in Thailand were identified as ST235 (Table 2). Among those, six clinical isolates were MBLs-producing P. aeruginosa which harbored bla_VIM_-₂ for 5 clinical isolates and only one isolate harbored bla_IMP_-_14a. All bla_IMP_-₁ producing isolates were identified as ST964. Moreover, both bla_IMP_-₆₅ harbored P. aeruginosa were characterized as ST111. Others MBLs harboring P. aeruginosa were identified as ST292, ST621 and ST1584 which were found only one clinical isolate each. Interestingly, most bla_VIM_-₂ carrying P. aeruginosa were identified as ST235 but one isolate was identified as ST1584. Among non-harbored MBL gene CR-MDR clinical isolates, ST235 was also identified as a dominant type. ST273 and ST1816 were also identified in this non-harbored MBLs group. It was noteworthy that ST235 was found as dominant ST (52.4%) in Thailand followed by ST964 (14.3%) and ST111 (9.5%). ST292, ST273, ST621, ST1584 and ST1816 were found only in one clinical isolate each.

Table 2

Multi-locus sequence typing allelic profiles and sequence types of 21 Pseudomonas aeruginosa clinical isolates collected across Thailand

Strains		ST/Pulsotype	MBLs	Region of origin	Antimicrobial susceptibility
Strains		ST/Pulsotype	MBLs	Region of origin	IPM	MEM	PIP	CAZ	CIP	GEN
No metallo beta-lactamases producing P. aeruginosa
	MPPA-051	235	Not detected	N	S	R	S	R	R	R
	MPPA-136	235	Not detected	NE	S	I	S	R	R	R
	MPPA-241	235	Not detected	C	S	I	R	R	R	R
	MPPA-328	235	Not detected	S	S	R	S	R	R	R
	MPPA-342	235	Not detected	S	S	R	S	R	R	R
	MPPA-222	273	Not detected	E	S	I	R	R	R	R
	MPPA-002	1816	Not detected	E	S	I	R	R	R	R
Metallo beta-lactamases producing P. aeruginosa
	MPPA-092	235	VIM-2	N	R	R	R	R	R	R
	MPPA-198	235/B	VIM-2	C	R	R	R	R	R	R
	MPPA-213	235	VIM-2	E	R	R	R	R	R	R
	MPPA-217	235/A	VIM-2	E	R	R	R	R	R	R
	MPPA-286	235/G	IMP-14a	S	R	R	R	R	R	R
	MPPA-409	235/C	VIM-2	NE	R	R	R	R	R	R
	MPPA-066	964/D	IMP-1	N	R	R	R	R	R	R
	MPPA-068	964	IMP-1	N	R	R	R	R	R	R
	MPPA-103	964/E	IMP-1	N	R	R	R	R	R	R
	MPPA-138	111	IMP-65	NE	R	R	R	R	R	R
	MPPA-155	111/F	IMP-65	NE	R	R	R	R	R	R
	MPPA-179	292	IMP-14a	C	R	R	R	R	R	R
	MPPA-263	621	IMP-13	C	R	R	S	R	R	R
	MPPA-135	1584	VIM-2	NE	R	R	R	R	R	R
	MPPA-215	ND/A	VIM-2	E	R	R	R	R	R	R
	MPPA-223	ND/A	VIM-2	E	R	R	R	R	R	R
	MPPA-240	ND	VIM-2	C	R	R	R	R	R	R
	MPPA-248	ND	VIM-2	C	R	R	R	S	I	S
	MPPA-294	ND/B	VIM-2	S	R	R	R	R	R	R
	MPPA-413	ND/C	VIM-2	NE	S	R	R	R	R	R
	MPPA-425	ND/C	VIM-2	NE	R	R	R	R	R	R
	MPPA-432	ND/C	VIM-2	NE	R	R	R	R	R	R
	MPPA-65	ND/D	IMP-1	N	R	R	R	R	R	R
	MPPA-69	ND/D	IMP-1	N	R	R	R	R	R	R
	MPPA-71	ND/D	IMP-1	N	R	R	S	R	R	R
	MPPA-98	ND/E	IMP-1	N	R	R	R	R	R	R
	MPPA-357	ND/C	IMP-1	S	S	I	S	R	R	R
	MPPA-150	ND/F	IMP-14a	NE	R	R	R	R	R	R
	MPPA-300	ND/G	IMP-14a	S	R	R	S	R	R	R
	MPPA-375	ND/C	IMP-14a	S	R	R	R	R	R	R
	MPPA-452	ND	IMP-14a	NE	R	R	S	R	R	R

S, susceptible; I, intermediate; R, resistant; IPM, imipenem; MEM, meropenem; PIP, piperacillin; CAZ, ceftazidime; CIP, ciprofloxacin; GEN, gentamicin; ST, sequence typing.

A-G, indicated similar PFGE fingerprinting; ND, no ST determined; N, northern region; NE, northeastern region; C, central region; E, eastern region; S, southern region.

Discussion

In our study, clinical isolates were phenotypic screened by paper disc diffusion, DDST and CDT. Many clinical isolates were interpreted as positive. However, only 20% were detected MBL genes. These might be affected by the obscure definition of DDST method which was the appearance of either an enhanced or a phantom zone between the antimicrobial agent and the inhibitor disk was considered a positive result and indicative of MBL production. Moreover, IntegronI was also found in most tested clinical isolates and always related with many antibiotic resistance mechanisms. These might indicate that those CR-MDR clinical isolates could carry many antibiotic resistance mechanisms such as, loss of OprD, multiple efflux pumps, or antibiotic inactivating enzymes and could be affected the phenotypic screening method. However, all positive phenotypic screened were further identified for specific type of MBLs.

Most prevalence MBLs belong to IMP- and VIM- type in P. aeruginosa. However, one of the most recent discovered MBLs, the NDM-type was also reported worldwide [20]. In our studies, we discovered bla_IMP_-₁, bla_IMP_-₁₃, bla_IMP_-_14a, bla_VIM_-₂ and a novel MBLs, bla_IMP_-₆₅. Previous report of VIM-type MBLs in Thailand were bla_VIM_-₂, bla_VIM_-₅ and bla_VIM_-₄₅ [21]. In this study, we found only bla_VIM_-₂ but these clinical isolates were collected from tertiary or university hospitals located in different parts of Thailand. This might indicate that bla_VIM_-₂ MBLs could be dominated VIM-type MBLs in Thailand.

Some previous reports showed a variety of IMP-type in Thailand including bla_IMP_-₁, bla_IMP_-₁₃, bla_IMP_-₁₄, bla_IMP_-_14a, bla_IMP_-₃₂ and bla_IMP_-₄₈ [21 22 23 24 25]. In this study, many IMP-type MBLs sush as bla_IMP_-₁, bla_IMP_-₁₃, bla_IMP_-_14a and bla_IMP_-₆₅ were identified. The results also indicated that bla_IMP_-₁ and bla_IMP_-_14a might be dominant types of IMP-MBLs in Thailand. Novel IMP-type MBLs, IMP-65, was demonstrated an amino acid changed from IMP-14 at the position 101 from alanine to threonine. There were also some IMP-type, bla_IMP_-₃₂ and bla_IMP_-₄₈, which presented similar sequences with bla_IMP_-₆₅ and bla_IMP_-₁₄. Moreover, bla_IMP_-₁₄, bla_IMP_-_14a, bla_IMP_-₃₂, bla_IMP_-₄₈ and bla_IMP_-₆₅ were never reported in any clinical isolate from other countries than Thailand. These might indicate that bla_IMP_-₁₄ and variants were spreading locally and also dominant in Thailand.

Acquired MBL genes were always found within class I integrons which usually carried other additional resistance genes and also known as a gene cassette. IntegronI is an important mechanism that bacteria use to transfer resistance mechanisms. Most class I integrons are always carried within transposons which help the bacteria to transfer antibiotic resistance gene from one to another [26]. In this study, we also found that most MDR P. aeruginosa carried integronI gene. Moreover, all MBLs harbored P. aeruginosa except one strain (96.6%) also carried integronI gene which was similar to many previous studies [27 28].

The primers used in this study were designed to target different alleles of IMP-type MBLs. All IMP-type MBLs in Thailand were detected by IMP-C primers designed to detect all previously report IMP-type MBLs in Thailand but it was unable to distinguished specific alleles of IMP-type MBLs in Thailand. From these results, this set of primers may be used as molecular confirmation test to detect dominant type of IMP-type MBLs type for epidemiology control in Thailand.

ST235 was identified as a dominant ST in Thailand. Moreover, we also found that ST964 was also presented as a significant ST type among MBL-harbored P. aeruginosa. However, this study was emphasized that ST235 was the dominant ST in Thailand when compared the results with previous studies [12 29]. Kim et al. studied dissemination of carbapenemase P. aeruginosa but only a few clinical isolates from Thailand were included and found only ST235 [12]. Another ST from Thailand is ST260 which was detected in P. aeruginosa harbored bla_IMP_-₁₄ [29].

ST235, ST111 and ST175 are high-risk clones which now concerned as majority of P. aeruginosa clinical isolates [6]. However, in Asian countries, only a few ST111 have been reported [8]. In this study, ST111 was identified but ST175 was undiscovered. ST111 were also carried the novel IMP-type MBL, bla_IMP-65. These finding was quite interesting since ST111 was occasionally found in Asia and bla_IMP-65 or similar IMP-types have never been reported in other places. Unfortunately, the information about those strains cannot be retrieved other than the location of the collection.

ST273, ST292, ST621, ST964, ST1584, and ST1816 have also never been reported in Thailand before. ST964, the second most ST in this study, was reported in Singapore and UK [30 31]. Those stains of ST964 in Singapore were also carried bla_IMP_-₁ as found in our study. ST292 was another ST which were reported in Asian countries, China and Korea [32 33]. However, there was no report on ST621, ST1584, and ST1816 in Asia. It was interesting that we found ST621 which was always carrying bla_IMP_-₁₃ as same as in many countries [34]. From these results, it might indicate the different dissemination trait from European countries which identified a lot of ST111 and ST175. However, the larger groups of samples may be required to gain more data.

It was noteworthy that antibiotic susceptibility in this study was determined by disc diffusion method according to the CLSI guideline. Therefore, some alternative drugs such as, colistin, were not examined due to unavailable breakpoints. In addition, only strains shared similar PFGE fingerprinting were selected as representative strains

In conclusion, this was the first report on bla_VIM_-₂ and bla_IMP_-₁ types which were highly presented MBL genes in Thailand. Moreover, the novel IMP type, bla_IMP_-₆₅ was also identified and dominated locally in MBLs, bla_IMP_-₁₄ and variants. ST235 was dominant among MBL-producing P. aeruginosa as well as no MBL-producing P. aeruginosa while each of ST964 and ST111 represented the clones of P. aeruginosa producing IMP-1 and IMP-65, respectively. Moreover, it is also the first report on many P. aeruginosa STs in Thailand: ST273, ST292, ST621, ST1584, and ST1816 which emphasized the dissemination trait difference of MBLs harboring P. aeruginosa in Thailand.

ACKNOWLEDGEMENT

The authors would like to thank all investigators and colleagues of the Department of Microbiology, Faculty of Pharmacy, Mahidol University. This work was financially supported by the Thailand Research Fund and Mahidol University under Grant No. TRG-5880129.

Notes

^{Conflict of Interest} No conflicts of interest.

^{Author Contributions}

Conceptualization: PK.
Data curation: PK, WY.
Formal analysis: PK, WY, MY.
Funding acquisition: PK.
Investigation: PK, WY.
Methodology: PK, MY, MTC.
Project administration: PK, WY.
Resources: PK, MY, MTC.
Software: PK, MY.
Supervision: PK.
Validation: PK, MY, MTC.
Visualization: PK, WY, MY.
Writing - original draft: WY.
Writing - review & editing: PK, MY, MTC.

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SUPPLEMENTARY MATERIAL

Supplementary Table 1

Detail of IMP-type genes of bacteria

TOOLS

ORCID iDs

Piyatip Khuntayaporn
https://orcid.org/0000-0003-3236-8240

Wariya Yamprayoonswat
https://orcid.org/0000-0001-7793-3819

Montri Yasawong
https://orcid.org/0000-0003-3298-0851

Mullika Traidej Chomnawang
https://orcid.org/0000-0002-3200-9259

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