Emergence of DIM-1 and KPC-1 Genes Associated Carbapenem-Resistant Pseudomonas Aeruginosa Isolates in Three Major Hospitals in Hanoi, Vietnam (2010-2015)


 Background: Multidrug-resistant bacteria including carbapenem resistant Pseudomonas aeruginosa are recognised as an important cause of hospital-acquired infections worldwide. To determine the molecular characterisation and antibiotic resistant genes associated with carbapenem-resistant P. aeruginosa. Methods: we conducted whole-genome sequencing and phylogenetic analysis of 72 carbapenem-resistant P. aeruginosa isolated from hospital-acquired infection patients from 2010 to 2015 in three major hospitals in Hanoi, Vietnam. Results: We identified three variants of IMP genes, among which IMP-15 gene was the most frequent (n= 34) in comparison to IMP-26 (n= 2) and IMP-51 (n=12). We observed two isolates with imipenem MIC >128mg/L that co-harboured IMP-15 and DIM-1 genes and seven isolates (imipenem MIC> 128mg/L) with KPC-1 gene from the same hospital. MLST data showed that sequence types (ST) of 72 isolates were classified into 18 STs and phylogenetic tree analysis divided these isolates into nine groups. Conclusion: Our results provide evidence that not only IMP-26, but other variants of IMPs like IMP-15 and IMP-51 genes and several STs (ST235, ST244, ST277, ST310, ST773 and ST3151) have been disseminated in health care settings in Vietnam. Also, we report the first finding in Vietnam that two isolates belonging to ST1240 and ST3340 harboured two important carbapenemase genes (IMP-15 and, DIM-1) and seven isolates belonging to ST3151 of P. aeruginosa carried the KPC-1 gene, which could be a potential cause of seriously restricted available treatment options in healthcare settings.


Background
Antibiotic resistance has taken centre stage as a global health issue that demands public attention and commands resources to understand where the international community will be placed in 2020-2025 [1]. Concerns have been raised due to the rapid emergence and spread of carbapenem-resistant Gram-negative bacteria resistant to the "last resort" antibiotic group in-hospital treatments. In addition, bacteria have been found to be resistant to colistin, which is recommended to be used as salvage treatment for infections caused by carbapenem-resistant bacteria [2;3] With the emergence of resistance against these drugs, there might be no effective antibiotic treatment for these bacteria in the next 5-10 years.
Multidrug-resistant Pseudomonas aeruginosa is recognized as an important cause of hospital-acquired infections and is listed among the WHO priority pathogens for research and development of new antibiotics [4;5]. This bacterium is highly adaptable to environmental uctuations including low-level antibiotic exposure and many antibiotic resistance mechanisms such as reduced membrane permeability, drug e ux pumps, and enzymatic inactivation have been found.
The spread of antibiotic resistance genes through mobile genetic factors greatly contributes to the formation of antibiotic resistant P. aeruginosa [6;7]. P. aeruginosa is very well known to have multiple resistance mechanisms at the same time, limiting treatment choices [8;9;10]. Epidemiological classi cation of P. aeruginosa using pulsed-eld gel electrophoresis (PFGE) has been used as the gold standard for molecular epidemiology to characterize and identify the risk of transmission and spread of P. aeruginosa outbreaks in hospitals [11]. However, this technique has limited discriminatory capacity, high cost, complex work ow and does not provide detailed information on the evolutionary background of P. aeruginosa. Currently, next-generation sequencing usage is becoming broader as it provides data not only on the genetic relatedness at higher resolution but also on resistance associated genes and their relatedness and thus more insights into antimicrobial resistant bacteria. With this technique, the relatedness and transmission of hospital isolates can be assessed and used to guide infection control interventions locally. Moreover, sequence and evolutionary data contribute to enhance the global picture of AMR genes and associated bacteria [12;13].
Southeast Asia is considered as a "hot spot" of antibiotic-resistant bacteria and P. aeruginosa has also been identi ed as a common cause of hospital-acquired infections in Vietnam [14;15]. According to statistics of the Center for Disease Dynamics, Economics and Policy (CDDEP) in 2016, 36% of P. aeruginosa isolates in Vietnam were resistant to carbapenems, ranking second only after India [14]. A study in one hospital in Hanoi -Vietnam reported a carbapenemase-ST235 P. aeruginosa carrying IMP-15, IMP-26 and IMP-51 genes [16]. Although the P. aeruginosa ST235 isolates were identi ed to play an important role of concerning in relation to hospital-acquired infections, the resistance mechanisms have not yet been clearly de ned, and the sequence types of P. aeruginosa associated with antibiotic resistance genes differ markedly among communities, hospitals, and countries [16;17;18;19]. Also, the results of our surveillance in 3 major hospitals in Hanoi between 2011 to 2015 showed that 11.5% (48/416) of carbapenem-resistant P. aeruginosa isolates carrying the IMP gene. That nding leads to some research questions needed to be addressed: (i) What variants of the IMP gene in the carbapenem-resistant P. aeruginosa isolates are circulating in healthcare settings in Vietnam, and whether they are similar to other variants, which have been reported in previous studies? (ii) What differences in molecular characterization of the IMP versus non-IMP of P. aeruginosa isolates? To address these questions, we conducted this study to determine the molecular characterization and antibiotic resistance genes associated with carbapenem-resistant P. aeruginosa isolated from three major hospitals in Hanoi between 2010 and 2015. The results provide more understanding of the genomic characteristics and the pro le of antibiotic resistance genes of P. aeruginosa, thereby help developing appropriate strategies for treatment and prevention.

Hospital settings and isolates
Isolates were sent from three hospitals (hospital A, B and C) with high capacity, located in centre of Hanoi, the capital city of Vietnam. Hospitals A and B are reference health care settings with over 600-bed capacity each, and include many specialties such as surgery, paediatric, intensive care unit (ICU), etc. Hospital C is the largest surgical centre in Vietnam with over 1500 beds that performs different elds of surgery such as abdominal surgery, gastroenterology & hepato-biliary surgery, paediatric surgery, and urology surgery. Demographic and basic clinical information of patients whose specimens were carbapenem-resistant P. aeruginosa positive was collected from clinical notes including age, gender, date of admission, clinical diagnosis, the origin of collected sample, isolated bacterial strains, and date of sample collection. Treatment and clinical outcome data were not available for this study.
From the collection of carbapenem-resistant P. aeruginosa isolates (n = 416) collected between August 2010 and December 2015, we selected all of 48 isolates carrying IMP gene (18 isolates from hospital A, 12 from B and 18 from C), and randomly selected 24 non-carrying IMP isolates (12 isolates from hospital A, 8 from B and 4 from C). All of 72 of carbapenem-resistant P. aeruginosa isolates in this study were resistant to at least one antibiotic in the carbapenem group for antibiotic susceptibility by using disc diffusion testing according to international guidelines [20].
Bacterial identi cation and susceptibility testing Isolates were con rmed by the MALDI Biotyper system (Brucker Daltonik, GmbH, Germany). Minimum inhibitory concentrations (MICs) of seven antibiotics which are commonly used in treatment for P. aeruginosa infections in Vietnam, including imipenem (IMP), ceftazidime (CAZ), cipro oxacin (CIP), gentamicin (GEN), amikacin (AMK) aztreonam (AZT) and colistin (CS) (Sigma-Aldrich) were performed by agar dilutions according to Clinical and Laboratory Standards Institute (CLSI) guidelines − 2018 [20]. The broth micro-dilution of colistin susceptibility testing was analysed according to the standard of the European Committee on Antibiotic Susceptibility Testing (EUCAST).

Whole genome sequencing of P. aeruginosa
To prepare whole-genome sequencing libraries, genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Libraries of P. aeruginosa strains were prepared using the Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA). Multiplexed paired-end sequencing was performed using the MiSeq Reagent V3 Kit (2 × 300 cycles) on an Illumina MiSeq instrument.

Bioinformatics analysis
Whole genome sequences were analyzed using an in-house bioinformatics pipeline, which runs on a Conda environment under Linux. Brie y, we used FastQC Version 0.11.8 for quality control of raw reads. Reads were trimmed of the adaptor sequences and were subsequently de novo assembled into contigs using SPAdes (3.9.0) with a pre-de ned Kmers set. Antimicrobial resistance genes were identi ed from the assembled contigs using the ABRicate program to query the Res nder database V2.1 [21] Multilocus sequence typing (MLST) was conducted from the Shovill-output contigs, screening seven housekeeping genes against the PubMLST database. Alleles were submitted to the PubMLST database to get the sequence type. A phylogenetic tree based on the core genome SNPs was constructed from WGS data of the 72 P. aeruginosa isolates using Parsnp 1.2 and IQ-TREE 1.5 [21;22].

Discussion
In this study we characterized the antibiotic resistant pro les of 72 carbapenem-resistant P. aeruginosa with and without IMPs genes collected from three major hospitals in Hanoi, Vietnam. The majority of carbapenem-resistant P. aeruginosa infection cases in this study were from pneumonia and VAP cases and ten cases were surgical site infection. These observations suggest that potential dissemination of carbapenem-resistant P. aeruginosa may occur at different departments in these hospitals, either by transferring between patients or through healthcare workers.
Our study found that P. aeruginosa carried different combinations of antibiotic resistance genes leading to a broadspectrum resistance to various antibiotics. Among 72 strains, 48 P. aeruginosa isolates carried three different IMP genes, similar to the IMP variants has been reported from several countries, including Mexico, Korean, Singapore and in Hanoi, Vietnam [16;17;18;23]. Our results showed the predominance of IMP-15 and IMP-51. These data supported the evidence that not only IMP-26, but other variants of IMP like IMP-15 and IMP-51 genes have been disseminated in health care settings in Vietnam.
The DIM-1 gene (Dutch imipenemase 1) was detected for the rst time in Vietnam in two P. aeruginosa isolates from hospitals A and B belonging to two differences sequence types: ST1420 and ST3440. Previous studies have shown that DIM-1 gene encodes a group of B metallo-beta-lactamase enzymes capable of lysis of carbapenem antibiotics that was discovered in integron class 1 genetic element (intl1) of Pseudomonas stutzeri in the Netherlands in 2007 [24], and in P. aeruginosa in India (5%-2010) and Sierra Leone (46.7%-2013) [25].
We also found seven P. aeruginosa isolates carrying KPC-1 in Vietnam belong to sequence type ST3151. The KPC encoding gene was previously reported in P. aeruginosa in China [26]. We also found a plasmid carrying KPC gene in Enterobacteriaceae clinical isolates from hospital A in 2010 and from other hospitals of Vietnam, which are currently being characterized, suggesting that the KPC-1 encoding gene of the P. aeruginosa strains in the study might be acquired from K. pneumoniae and E. coli through conjugation. Colistin is the only effective antibiotic in some cases of P. aeruginosa resistance to all tested antibiotics, even carbapenem. However, the emergence of colistin-resistant strains is considered a great threat for patients with severe infections [28;29;30;31]. Our study found that 11.1% P. aeruginosa isolates were resistant to colistin compared to 7% (3%-13%) data of CDDEP collected from the Vietnam Resistance Project (VINARES) [14]. This result indicates that Vietnam is one of the countries having a high rate of colistin resistance. Our nding suggests a cautious consideration of using colistin in treatment for P. aeruginosa in clinical practices.
The MLST data showed a high diversity of P. aeruginosa isolates. The 72 isolates were grouped into 18 different sequence types, seven of these STs (ST235, ST360, ST310, ST357, ST277, ST773 and ST2166) have also been reported in Vietnam [16;19;32;33]. We also showed the clustering of ST following the hospitals. Particularly, the fact that ST360 mainly found in hospital C and ST3151 was predominant in hospital A strongly suggest that hospital transmission may have occurred. Core genome phylogenetic and STs of the isolates were also in the same group. Some high-risk STs were found within a hospital (ST360) or in different hospitals (ST235, ST244, ST277, ST 340, ST357) and in different years (Fig. 2, Fig. 3). Interestingly, ST3151 carries different carbapenem genes: KPC-1 (n = 7), IMP-15 (n = 1), and one strain only carries OXA-50, suggested that the KPC-1 positive-P. aeruginosa strains in the study might be acquired from K. pneumoniae and E. coli through conjugation transfer as mentioned above. These ndings suggest that these STs have been possibly disseminated in health care settings in Vietnam and new STs could be formed under selective and antibiotic pressure.
Our study has several limitations. Firstly, isolates might not represent for carbapenem-resistant P. aeruginosa in health care settings in Vietnam. Secondly, we were unable to assess the clinical signi cance of carbapenem-resistant P. aeruginosa regarding to antibiotic treatment and outcome. Therefore, we propose that future studies should incorporate clinical data to obtain a better understanding of characterization of P. aeruginosa infections in Vietnam.
Despite its limitations, our study shows the high diversity and different level and mechanisms carbapenem resistance among hospital P. aeruginosa isolates. Our data supports the evidence that not only IMP-26 but other variants of IMPs like IMP-15 and IMP-51 genes and several STs have been disseminated in health care settings in Vietnam. We also rstly reported the ST1420 and ST3340 which co-harboured IMP-15 + DIM-1, and seven ST3151 carrying KPC genes in Vietnam, which may cause a seriously restricted available treatment option in healthcare settings. Core genome phylogenetic tree of the 72 P. aeruginosa isolates of the three hospitals associated with sequence types and antibiotic resistant genes. The shapes stand for hospitals ; colors of the shapes stand for collection years of isolates; each red square indicates the presence of AMR genes in isolates.