Expansion of KPC-producing Enterobacterales in four large hospitals in Hanoi, Vietnam

Highlights • 122 (20.4%) of 599 carbapenem-resistant Enterobacterales (CRE) isolates carried blaKPC genes.• Predominance of ST15 K. pneumoniae, whereas E. coli presented more diverse sequence types.• blaKPC-bearing plasmids were diverse in size.• Three different models of genetic context of blaKPC-2.• Hypothesis of circulation of resistant bacteria and transmission among hospitals.


a b s t r a c t
Objectives: : The incidence of carbapenem resistance among nosocomial Gram-negative bacteria in Vietnam is high and increasing, including among Enterobacterales. In this study, we assessed the presence of one of the main carbapenemase genes, bla KPC , among carbapenem-resistant Enterobacterales (CRE) from four large hospitals in Hanoi, Vietnam, between 2010 and 2015, and described their key molecular characteristics. Methods: : KPC-producing Enterobacterales were detected using conventional PCR and were further analysed using S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and whole-genome sequencing (WGS) for sequence typing and genetic characterisation. Results: : bla KPC genes were detected in 122 (20.4%) of 599 CRE isolates. bla KPC -carrying plasmids were diverse in size. Klebsiella pneumoniae harbouring bla KPC genes belonged to ST15 and ST11, whereas KPCproducing Escherichia coli showed more diverse sequence types including ST3580, ST448, ST709 and ST405. Genotypic relationships supported the hypothesis of circulation of a population of 'resident' resistant bacteria in one hospital through the years and of transmission among these hospitals via patient transfer. WGS results revealed co-carriage of several other antimicrobial resistance genes and three different genetic contexts of bla KPC-2 . Among these, the combination of IS Ecp1 -bla CTX-M and IS Kpn27 -bla KPC -IS Kpn6 on the same plasmid is reported for the first time.

Introduction
The incidence of carbapenem resistance among nosocomial pathogens in Vietnam is high and increasing, especially among Escherichia coli and Gram-negative 'ESKAPE' organisms ( Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.). Data from a nationwide hospital surveillance network in 2016-2017 showed that rates of carbapenem resistance among K. pneumoniae, E. coli and Enterobacter spp . were 29%, 11% and 27%, respectively [1] . A recent study from Vietnam including more than 2200 patients admitted to 12 hospitals throughout the country during 2017 and 2018 reported that 52% of patients were colonised with carbapenem-resistant Enterobacterales (CRE) [2] . Since the first NDM-1-producing K. pneumoniae in Vietnam was isolated from the urinary tract of a 62-year-old hospitalised patient in 2010 [3] , most class A, B and D carbapenemases in Enterobacterales have been reported from Vietnam [4][5][6] . In the southern part of Vietnam, carbapenem-resistant K. pneumoniae clinical isolates producing various carbapenemases such as KPC-2, NDM-1, NDM-4 and OXA-48 have been described [ 6 , 7 ]. Studies from three hospitals in Hanoi, in the northern part of Vietnam, detected diverse variants of carbapenemase genes such as KPC-2, KPC-3, KPC-4, NDM-1, IMP-4, IMP-79, VIM-1 and OXA-48 in Enterobacterales [ 4 , 5 , 8 , 9 ]. Data on the molecular characteristics of carbapenemases from carbapenemase-producing Enterobacterales (CPE) in clinical isolates in Vietnam, a lower-middle income country with a high and increasing burden of antimicrobial resistance and hospital-acquired infections, are still sparse. Here we present the phenotypic and molecular characteristics of KPC-producing CPE isolates from four major hospitals in Hanoi between 2010 and 2015 in order to gain a better understanding of the circulation of CPE in Vietnam and to compare this with local, regional and global data to add to the current knowledge base . Our results will contribute to outline a larger picture of CPE in Vietnam and will serve as important scientific information for government action plans on antibiotic resistance control.

Study sites and sample collection
We prospectively collected CRE isolates from four large hospitals including Saint Paul (A), Thanh Nhan (B), Viet Duc (C) and 108 Military Central Hospital (D) located in the centre of Hanoi, Vietnam. All four are public hospitals; three are general hospitals (A, B and D) and C is a specialised hospital for surgery. A and B are city hospitals with a 600-bed capacity, while C and D are central hospitals with a greater than 10 0 0-bed capacity. Demographic and basic clinical information of patients whose specimens were positive for CRE were collected from clinical notes and included age, sex, date of admission, clinical diagnosis, origin of collected sample, date of sample collection and culture results. Treatment and clinical outcome data were not available for this study.
CRE isolated from clinical specimens were tested for antimicrobial susceptibility at the four sites by the disk diffusion method ac-cording to Clinical and Laboratory Standards Institute (CLSI) guidelines [ 10 , 11 ]. Microbiology laboratories in the four hospitals were requested to collect and send all bacterial isolates resistant to at least one carbapenem to the National Institute of Hygiene and Epidemiology for further characterisation ( n = 599, including 179 isolates from hospital A, 87 from hospital B, 95 from hospital C and 238 from hospital D).

Antimicrobial susceptibility testing and detection of antimicrobial resistance genes
Minimum inhibitory concentrations (MICs) were determined centrally by the agar dilution method for imipenem, meropenem, cefotaxime, ceftazidime and ciprofloxacin and by microdilution for colistin (Sigma-Aldrich) according to CLSI and European Committee on Antimicrobial Susceptibility testing (EUCAST) guidelines [ 11 , 12 ]. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control strains.
Collected isolates were screened for four common carbapenemase genes, including bla KPC , bla NDM-1 , bla IMP and bla OXA-48 , as well as three other common β-lactamase-encoding genes ( bla TEM , bla SHV and bla CTX-M ) as described previously [4] . Resulting amplicons were sequenced using conventional Sanger sequencing.

Multilocus sequence typing (MLST)
MLST was done using PubMLST for all bla KPC -positive isolates ( n = 122) [13] . Briefly, seven housekeeping genes were amplified by PCR, sequenced and compared with the sequences submitted to the MLST database to determine the sequence type (ST).

Whole-genome sequencing (WGS) and characterisation of the genetic environment of bla KPC genes
A subset of bla KPC -carrying CPE isolates ( n = 69) was selected for further analysis including representatives of different hospitals, collection years, departments and sample types. Genomic DNA libraries of selected strains were prepared for WGS using a Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer's instructions. Then, 300-bp paired-end sequencing was performed on an Illumina MiSeq platform (MiSeq Reagent Kit v3; 600 cycles). Raw sequence reads were de novo assembled into contigs using SPAdes v.3.9.0 with predefined Kmers set. Antimicrobial resistance genes were identified using ResFinder v.2.1, and MLST profiles were generated using the Phylogenetic trees based on the core genome single nucleotide polymorphisms (SNPs) were constructed from WGS data of the 60 bla KPC -carrying K. pneumoniae and 9 bla KPC -carrying E. coli isolates using Parsnp 1.2 and IQ-TREE 2.0 [ 15 , 16 ].

Statistical methods
Isolates and patient data were analysed in Microsoft Excel 2017 (Microsoft Corp., Redmond, WA, USA) using descriptive statistics as appropriate. Of the 599 CRE isolates, 122 (20.4%) were positive for bla KPC , including 13 E. coli (10.7%) and 109 K. pneumoniae (89.3%). No bla KPC gene was detected in the other bacteria. Hospital A contributed the largest number of bla KPC -positive isolates ( n = 101, accounting for 82.8%), followed by hospital B ( n = 9; 7.4%), hospital D ( n = 8; 6.6%) and hospital C ( n = 4; 3.3%) ( Table 1 ). The first detected bla KPC -positive bacterium was K. pneumoniae , isolated from bronchial fluid of a ventilated patient in the intensive care unit (ICU) of hospital B on 16 January 2010.

Antimicrobial resistance gene profile, sequence typing and genotypic relationship
WGS data revealed the resistance profile of bla KPC -harbouring isolates to a wide range of antibiotics ( Table 4 ). All isolates carried genes conferring resistance to at least three different antibiotic categories, but with considerable variation in the resistanceconferring elements carried. Indeed, two resistance genes ( fosA and dfrA ) were present in all K. pneumoniae isolates but not all E. coli isolates. On the other hand, the mph(A) gene was observed in all E. coli but only a few K. pneumoniae isolates ( Figs 1 and 2 ). Notably, one K. pneumoniae isolate carried resistance genes for all 10 investigated antibiotic categories. All 60 (100%) bla KPC -carrying K. pneumoniae isolates carried resistance genes against β-lactams, fosfomycin and trimethoprim. Resistance elements to aminoglycosides and quinolones were also detected in high proportions ( ≥95%) among K. pneumoniae isolates; however, few strains had genotypic resistance to macrolides. In E. coli , all nine (100%) bla KPCcarrying isolates carried genes encoding resistance to aminoglycosides, β-lactams, tetracyclines and macrolides. One E. coli isolate carried genes conferring resistance to quinolones.
The core genome phylogenetic tree of 60 bla KPC -carrying K. pneumoniae isolates presented only three genotypic groups: two groups had ST15 and one group had ST11 ( Fig. 1 ). The largest ST15 lineage contained all isolates from three hospitals (A, B and C) collected during 2010 and 2015. Interestingly, two resistance gene patterns were observed in this lineage originated from isolates from the three hospitals through the years. In contrast, strains collected from hospital D constituted two separate ST15 and ST11 groups, sharing a quite similar resistance gene profile.
KPC-producing E. coli had more diverse sequence types, including ST3580, ST448, ST709 and ST405 ( Table 5 ), corresponding to four genotypic groups in its core genome phylogenetic tree ( Fig. 2 ). The resistance profile was similar among E. coli strains belonging to the same sequence type.

Characterisation of bla KPC -carrying plasmids and genetic environment of bla KPC
Randomly selected bla KPC -positive isolates ( n = 24) from the four hospitals were subsequently analysed by S1-PFGE and South-             ern blotting for bla KPC , showing that most (21/24) bla KPC genes were plasmid-borne ( Fig. 3 ). Interestingly, the size of bla KPCcarrying plasmids in hospital A and B was similar ( ∼30 kb). In contrast, bla KPC -positive K. pneumoniae in hospitals C and D carried plasmids different in size: ∼170 kb and ∼55 kb, respectively. Two bla KPC -positive E. coli isolates and one K. pneumoniae isolate did not hybridise. IncFIB(K), IncN and IncFIIK were predominant plasmid types among bla KPC -carrying plasmids. Analysis of the genetic environment of the bla KPC-2 gene revealed various genetic contexts of bla KPC-2 ( Fig. 4 ) in different plasmids. Tn 4401b isoform harbouring the bla KPC-2 gene was present in several E. coli and K. pneumoniae isolates carrying 55-bp or 30-

Discussion
Since their first detection in the USA [17] , bla KPC -carrying plasmids have rapidly spread across countries and continents and the bla KPC-2 gene was recently described in several hospitals in Vietnam [ 4 , 6-9 ]. Here we report the widespread distribution of bla KPC -producing Enterobacterales from samples collected from patients admitted to four large hospitals between 2010 and 2015 in Hanoi, Vietnam, with the first isolate detected in early 2010. It is possible there was already undetected circulation prior to 2010. bla KPC genes were detected in 122 (20.4%) of 599 CRE isolates.
The most dominant sample type was bronchial fluid usually collected from mechanically ventilated patients, which are commonly found to be associated with nosocomial infections [18] . Genes conferring resistance to different antibiotic categories were also observed in other CRE isolated in Vietnam [ 5 , 8 ]. This shows the increasing number of diverse resistant strains in hospital settings in Vietnam, which pose a great challenge for doctors in efficient antibiotic selection.
The predominance of ST15 in the four hospitals was similar to other published data in Vietnam and Asian countries including China, which shares a > 10 0 0 km border and trading and tourism activities with Vietnam [ 6 , 8 , 19 , 20 ]. ST15 K. pneumoniae has been reported worldwide as a clone carrying multiple carbapenemase genes, which was also observed in other studies in Vietnam [ 8 , 21 ]. Carbapenemase-producing ST15 K. pneumoniae was first reported in Vietnam in samples collected between 2014-2015 [6] ; however, in our study they were found in samples collected since 2010, facilitating the hypothesis of their presence in Vietnam hospitals prior to 2010. All bla KPC -carrying K. pneumoniae isolated from hospital A throughout the study period belonged to ST15, suggesting that they were resident flora. Although ST11 was reported among carbapenem-resistant K. pneumoniae in Vietnam previously [ 22 , 23 ], it was detected in only two isolates in the current study. This sequence type has also been associated with nosocomial outbreaks in several countries, especially in China [ 21 , 24 , 25 ]. Isolates from hospitals A, B and C belonged to the same lineage, supporting the hypothesis of exchange of bacteria and plasmids between these hospitals via patient transfer. Hospital D served a different targeted patient population and did not transfer patients with the other hospitals, which might explain the finding that resistant strains collected here evolved in their own ways forming two separate lineages.
Plasmid types carrying bla KPC genes found in this study were diverse and similar to those in many countries such as the UK, USA and China [26][27][28] . Four plasmid sizes were detected in this study that differed from the 150-kb bla KPC -carrying plasmid observed previously in Vietnam [6] . Notably, bla KPC -carrying plasmids isolated from hospital D all had the same size ( ∼55 kb) but belonged to two different ST groups (ST11 and ST15). This evidence supports the independent existence of bla KPC -carrying plasmids and, together with the diversity of sizes of bla KPC -carrying plasmids, shows the possibility of plasmid transmission across bacterial strains and species.
Regarding the gene context models of bla KPC-2 , the Tn 4401 isoform is endemic in many countries, whereas it is infrequently observed in China. Instead, IS Kpn27 -bla KPC -IS Kpn6 within the Tn 3 transposon frame accounted for the majority of isolates from China but not in other countries, which was also found in our study (Model II) [ 21 , 29 ]. This model was not only observed in Enterobacterales such as E. coli, K. pneumoniae and Citrobacter freundii but was also detected in P. aeruginosa [29][30][31] , showing the possibility of plasmid transmission between different species and genera of bacteria. All isolates carrying this model in our study showed high resistance to carbapenems (MIC ≥ 8 μg/mL), except for one isolate maintaining susceptibility to imipenem.
Model III with the combination of IS Ecp1 -bla CTX-M and IS Kpn27bla KPC -IS Kpn6 in the same plasmid ( Fig. 4 ) has not been reported before. However, the co-existence of one plasmid carrying IS Ecp1bla CTX-M and one plasmid carrying IS Kpn27 -bla KPC -IS Kpn6 in one isolate was reported from China in 2010 [32] . This suggests that a recombination event occurred bringing these two structures into one plasmid during the evolution of the isolate/mobile gene element. Interestingly, MIC results showed that this combination confers only weak resistance to carbapenems but still strong resistance to cefotaxime. Insertion of IS Ecp1 -bla CTX-M into the IS Kpn27 -bla KPC -IS Kpn6 frame might affect the phenotype of carbapenem resistance.
This study has several limitations. We do not have full hospital denominators and only a limited amount of metadata were collected, and we do not know patient treatment outcomes. Therefore, we were unable to draw further conclusions on the epidemiological characteristics (such as burden of disease, distinction of community-acquired and hospital-acquired isolates, patient-topatient and environmental persistence, commensal and pathogenic bacteria) of bla KPC -expressing Enterobacterales in these hospitals.
In conclusion, we describe the widespread presence of bla KPCexpressing Enterobacterales in four large hospitals in Hanoi, Vietnam, since 2010, which may have started earlier, along with their resistance patterns, sequence types, genotypic relationship, plasmid sizes and genetic context. The spread of these carbapenemaseproducers adds an additional challenge to the treatment of diseases caused by these common bacteria with a very extensive expression of genes conferring additional resistances.
Our study also provides evidence for the likelihood of KPCproducer circulation among three of the four hospitals as well as the possibility of plasmid transmission across bacterial strains and species. Data from this study contribute to a more comprehensive picture of the antimicrobial resistance situation in hospitals in Hanoi in the context of overcrowding and lack of hospital infection control programmes.

Ethical approval
The samples used in this study were taken from the Isolate Bank of the National Institute of Hygiene and Epidemiology (NIHE). This study is part of the main project 'Assessing the impact and burden of antimicrobial resistance in Vietnam, genomic characterization and risk factors related to antimicrobial resistance of common bacteria in Vietnam', which was approved by the institutional review board (IRB) of NIHE [IRB code IRB-VN01057-38/2016]. Individual informed consent was waived because of the retrospective nature of the work and because no personal identifiers were collected.

Declaration of Competing Interest
None declared.