An Outbreak of KPC-Producing Klebsiella pneumoniae Linked with an Index Case of Community-Acquired KPC-Producing Isolate: Epidemiological Investigation and Whole Genome Sequencing Analysis

Abstract

Aims:

A hospital outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPN) linked with an index case of community-acquired infection occurred in an urban tertiary care hospital in Seoul, South Korea. Therefore, we performed an outbreak investigation and whole genome sequencing (WGS) analysis to trace the outbreak and investigate the molecular characteristics of the isolates.

Results:

From October 2014 to January 2015, we identified a cluster of three patients in the neurosurgery ward with sputum cultures positive for carbapenem-resistant KPN. An epidemiological investigation, including pulsed-field gel electrophoresis analysis was performed to trace the origins of this outbreak. The index patient's infection was community acquired. Active surveillance cultures using perirectal swabbing from exposed patients, identified one additional patient with KPC-producing KPN colonization. WGS analyses using PacBio RSII instruments were performed for four linked isolates. WGS revealed a genetic linkage of the four isolates belonging to the same sequence type (ST307). All KPN isolates harbored conjugative resistance plasmids, which has bla_KPC-2 carbapenemase genes contained within the Tn4401 “a” isoform and other resistance genes. However, WGS showed only three isolates among four KPC-producing KPN were originated from a common origin.

Conclusions:

This report demonstrates the challenge that KPC-2-producing KPN with the conjugative resistance plasmid may spread not only in hospitals but also in community, and WGS can help to accurately characterize the outbreak.

Introduction

Klebsiella pneumoniae (KPN) is a member of the Enterobacteriaceae family, commonly found in the normal microbiota of human skin, and is also an important opportunistic pathogen.¹ Klebsiella pneumoniae carbapenemase (KPC)-producing KPN has emerged as a threatening nosocomial pathogen worldwide.² KPC enzymes are class A carbapenemases that hydrolyze carbapenems³ and also other β-lactam antibiotics. Individuals with infections caused by this pathogen have high mortality rates; they are difficult to treat because KPC-producing KPN is resistant to the majority of antimicrobial agents, including carbapenems.^4,5 The first KPC-1-producing KPN isolate was identified in 1996 in the United States.⁶ Since KPC-producing KPN was first reported in South Korea in 2010,⁷ several hospitals have experienced outbreaks caused by this pathogen.^8,9 Most KPC-producing KPN are nosocomial pathogens; however, community-acquired infection has also been reported.¹⁰

The first outbreak of KPC-producing KPN from South Korea in 2011 was associated with KPN ST258,⁸ and the sporadic emerging KPC-producing KPN belonged to ST258 and ST11.^7,11 The well-known KPC-producing clone in a global level is KPN ST258,^12,13 however, the National Laboratory Surveillance System for carbapenemase-producing Enterobacteriaceae (CPE) by the Korea Center for Disease Prevention and Control showed that the foremost South Korean KPC producer was KPN ST307 clone and the ST11 was followed by presenting half the prevalence of ST307.^14,15 The South Korean ST307 clone are assumed to be close to the Italian clone in an evidence of most KPC-producing KPN ST307 coproduced CTX-M enzyme.^14,16 The second-most disseminated KPN ST11 is one of the main KPC-producing clones in China.¹⁷

Antibiotic susceptibility profiles or molecular genotyping methods such as pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing (MLST) have been useful for studying outbreaks and for evaluating associations between bacterial strains. However, detection of KPC-producing bacteria, relying only on susceptibility testing is challenging due to the heterologous expression of β-lactam resistance.⁵ The phylogenetic tools applied to bacterial whole genome and core genome single nucleotide polymorphism (SNP) analyses have become an integral part of the epidemiological monitoring of multidrug-resistant pathogens to distinguish outbreak from nonoutbreak strains in hospital settings.¹⁸ The most important function of the KPN genome framework in recent years has been the identification of clonal groups and antimicrobial resistance determinants, including acquired genes and common mutations.¹⁹ As a consequence, transmission events can be accurately reconstructed and the appropriate measures for controlling hospital infection can be improved.

We recently experienced a hospital outbreak of KPC-producing KPN linked to a community-acquired index case. Therefore, we performed an outbreak investigation and whole genome sequencing (WGS) analysis to trace the outbreak and investigate the molecular characteristics of the isolates.

Materials and Methods

Study setting

This outbreak investigation was performed at a 2,500-bed tertiary care medical center in Seoul, South Korea between October 2014 and February 2015. The facility is a university hospital providing services in a wide range of specialties, and has a total of 105 intensive care unit (ICU) beds (53 medical/surgical, 22 dedicated to cardiothoracic surgery and cardiology, and 30 for neurosurgery). We experienced the first KPC-producing KPN outbreak at this institute during the aforementioned study period.

Microbiology and molecular tests

Bacterial identification and antimicrobial susceptibility tests were performed using the VITEK 2 system (bioMerieux, Hazelood, MO) and the disk-diffusion method. The results were interpreted according to the 2014 guideline of Clinical Laboratory Standards Institute.²⁰ Resistance to carbapenem was defined as a minimum inhibitory concentration (MIC) to imipenem or meropenem of ≥4 μg/mL or a disk diffusion of ≤19 mm.

The presence of carbapenemase was confirmed by PCR and sequencing for bla_IMP, bla_VIM, bla_NDM, bla_KPC, and bla_OXA using previously reported primers.^8,9,21,22 Determined nucleotide sequences were compared with sequence databases using BLAST.^*

PFGE was performed to determine the genetic relatedness between the isolates, as previously described.⁹ XbaI-digested genomic DNA was prepared, and DNA fragments were separated for 20 hr at 6 V/cm at 11°C using a CHEF-DRII System (Bio-Rad, Hercules, CA) with initial and final pulse times of 0.5 and 30 sec, respectively. Banding patterns were analyzed using the UVIband/Map software (UVItech Ltd., Cambridge, United Kingdom) to generate a dendrogram based on the unweighted pair group method using arithmetic averages from the Dice coefficient.

Epidemiological investigation

From October 2014 to January 2015, we identified a cluster of three patients in the neurosurgery ward with sputum cultures positive for carbapenem-resistant KPN. The infection control office performed the outbreak investigation. A relevant case was defined as the occurrence of KPC-producing KPN isolated from any specimen collected during the outbreak period (October 2014 through March 2015). Active surveillance culturing of perirectal swabs from patients potentially exposed to the three afflicted patients was performed to prospectively identify additional cases. The electronic medical record system alerts the infection control practitioner in real time to any Gram-negative bacilli reported as carbapenem resistant; this triggered contact precautions per standard practice at the hospital. Data on patient demographic characteristics, clinical characteristics, transfer history within the facility, invasive procedures, and involved health care workers were collected from the electronic medical records.

Interventions

Active surveillance cultures (ASCs) for the potentially exposed three patients were performed to prospectively identify additional cases with KPC-producing KPN using perirectal swabbing. We actively surveilled all patients who shared the same wards or ICUs, as well as those who were cared for by the same health care workers. A total of 37 patients were subject to ASCs. We did not perform cultures from environmental surfaces.

WGS analysis

Genomic DNA was extracted from the strains using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Next, the DNA was sheared using a g-TUBE™ apparatus (Covaris, Inc., Moburn, MA) and the fragments were purified by using 0.45 × of the final volume of washed Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA). Genomic library construction (10-kb libraries for all samples and additional 20-kb one for ST307PT01) and single-molecule real-time (SMRT) sequencing using a PacBio RSII instrument (P6-C4 chemistry) were performed at ChunLab (Seoul, Korea). Three SMRT cells (patient 1) or two cells (the other patients) were used for the sequencing. HDF5 files were processed using the RS_HGAP_Assembly.2 protocol in SMRT Analysis 2.3.0. Polished contigs were further processed using the Circlator tool,²³ and additional rounds of Quiver correction (RS_Resequencing.1 protocol in SMRT Analysis) were applied to eliminate residual errors. Accession numbers (CP022916–CP022926) for the complete chromosome sequences and plasmids are shown in Supplementary Table S1. All PacBio raw sequencing data files are available from SRA under the project accession number SRP115560 (PRJNA396106).

Genome annotation was performed using Prokka v1.11 and NCBI Prokaryotic Genome Annotation Pipeline.^24,** Replicase genes from the putative plasmids were classified using the PlasmidFinder online tool. SNPs between ST307PT01 and the other strains were found using Snippy and SNP-dist.

MLST was identified using whole genome sequences in the MLST database.^† Antimicrobial resistance genes were identified using ResFinder and a cutoff of 80% similarity. Whole genome alignment and visualization were carried out using Mauve v2.4.0, MUMmer v3.23, or cross_match. For the detection of differentially repeated segments from the plasmid, Gepard was used. Approximately maximum likelihood phylogenetic relationship were inferred using FastTree 2²⁵ based on core gene alignment calculated using the Roary tool.²⁶ Strains for the comparative genomic analysis were chosen among ST11, ST258, and ST307 strains isolated all over the world: HS11286 (GCF_000240185.1),²⁷ YMC 2013/7/B3993 (GCA_001548535.1),²⁸ GSU10-3 (GCF_003584585.1),²⁹ TR191 (GCF_001613565.1),³⁰ KPNIH1 (GCF_000281535.2),³¹ NJST258_1 (GCF_000598005.1),³² 236/11 (SRA ERS1477139),³³ KP48 (GCF_001566595.1),³⁴ KH-43 (GCF_002166965.1),³⁵ H150820806 (GCF_002167035.1),³⁵ and CFSAN045564 (GCF_001720845.1).³⁶ For strain 236/11, Illumina sequencing reads were downloaded and de novo assembled using SPAdes v3.11.1, followed by Prokka genome annotation.

Results

Epidemiological results

The index case (patient 1) was a 74-year-old man who visited the emergency room (ER) in October, 2014. He had no history of health care facility use, including any admission and surgery before the current ER admission. He had experienced a sudden onset of headache, nausea, and vomiting, and was diagnosed with intracranial hemorrhage. On the day of ER admission, he underwent a surgery for decompressive craniectomy and hematoma removal. A sputum culture obtained before the surgery due to pneumonic consolidation observed on his chest radiography image was positive for KPN. The isolate was resistant to meropenem (MIC >16 μg/mL) and contained bla_KPC as verified by PCR, and sequencing of carbapenemase. Patient 1 was transferred from an open bed in the neurovascular care unit (NCU) to an isolation room within the same unit in October, 2014, shortly after confirmation of carbapenem-resistant KPN, and contact precautions were then applied. He was transferred to a general ward in December, 2014 and discharged from the hospital in March, 2015. KPC-producing KPN was repeatedly isolated from his stool culture during his hospital stay.

Patient 2 was a 72-year-old man who was admitted after an ER visit for the evaluation and treatment of quadriplegia caused by the traumatic cervical fifth vertebral fracture in September, 2014. Total cervical corpectomy and decompression were performed. He stayed in the NCU in October, 2014, for the treatment of pneumonia, and was then transferred to the pulmonology department's ICU. KPC-producing KPN was isolated from a sputum culture performed at the pulmonology general ward (ward 2) in November, 2014. KPC-producing KPN was repeatedly isolated from the stool culture during his hospital stay.

Patient 3 was an 80-year-old man who was admitted for comprehensive rehabilitation treatment for hemiplegia caused by a right middle cerebral artery infarction in September, 2014. He was transferred from the rehabilitation general ward to the medical ICU to treat respiratory failure with pneumonia. Afterward, he stayed in the pulmonology general ward (ward 2) from November, 2014 onward. KPC-producing KPN was isolated from a sputum culture performed in ward 1 in January, 2015.

On February 2015, ASCs for potentially exposed patients were performed to detect any KPC-producing KPN using perirectal swabs. The subjects included in the active surveillance included all patients in the same wards (ward 1, 2, and 3) or ICUs (NCU and medical ICU) as well as all patients who were cared for by the same health-care workers. This ASC identified a patient (patient 4) who carried KPC-producing KPN. He was an 80-year-old man treated for quadriplegia caused by subdural hygroma and aspiration pneumonia, and had stayed in ward 2 in January, 2015. Table 1 shows the clinical characteristics of the four patients with KPC-producing KPN. Figure 1 shows the timeline of the patients' admission and potential transmission opportunities. No additional patients with the same KPC-producing KPN clone were identified up to September 2015.

FIG. 1.

Diagram describing a timeline of patients' admission and potential transmission opportunities. ICU, intensive care unit; NCU, neurovascular care unit.

Table 1.

Characteristics of the Four Patients and Minimal Inhibitory Concentrations of the Klebsiella pneumoniae Carbapenemase-Producing Klebsiella pneumoniae Isolates

Patient characteristic	Patient 1	Patient 2	Patient 3	Patient 4
Age/Sex	74/M	72/M	80/M	80/M
Nursery history	No	No	Yes	No
Underlying	Intracranial hemorrhage	Quadriplegia Traumatic vertebral fracture	Hemiplegia Right middle cerebral artery infarction	Quadriplegia Subdural hygroma
Surgery	Decompressive craniectomy, hematoma removal	Total cervical corpectomy and decompression	None	None
Diagnosis	Pneumonia	Pneumonia	Pneumonia	Pneumonia
Type of specimen	Sputum	Sputum	Sputum	Rectal swab
Isolation date	10/2014	11/2014	1/2015	2/2015
Prior antibiotics use	No	Yes	Yes	Yes
Outcome	Survived	Survived	Deceased	Survived

Antimicrobial agent	Minimum inhibitory concentration (μg/mL)
Antimicrobial agent	ST307PT01	ST307PT02	ST307PT03	ST307PT04
β-Lactams
Penicillin
Ampicillin	>32 (R)	>32 (R)	>32 (R)	>32 (R)
Ampicillin/sulbactam	>32 (R)	>32 (R)	>32 (R)	>32 (R)
Piperacillin/tazobactam	>128 (R)	>128 (R)	>128 (R)	>128 (R)
Cephalosporin
Cefazolin	>64 (R)	>64 (R)	>64 (R)	>64 (R)
Cefoxitin	16 (I)	16 (I)	64 (R)	64 (R)
Cefotaxime	>64 (R)	4 (R)	8 (R)	8 (R)
Ceftazidime	>64 (R)	16 (R)	16 (R)	16 (R)
Cefepime	>64 (R)	2 (S)	2 (S)	2 (S)
Aztreonam	>64 (R)	>64 (R)	>64 (R)	>64 (R)
Carbapenem
Meropenem	>16 (R)	>16 (R)	>16 (R)	>16 (R)
Ertapenem	>8 (R)	>8 (R)	>8 (R)	>8 (R)
Aminoglycoside
Gentamicin	>16 (R)	<1 (S)	<1 (S)	<1 (S)
Amikacin	<2 (S)	<2 (S)	<2 (S)	<2 (S)
Levofloxacin	>8 (R)	>8 (R)	>8 (R)	>8 (R)
Cotrimoxazole	>320 (R)	>320 (R)	>320 (R)	>320 (R)
Tigecycline	2 (S)	2 (S)	>8 (R)	>8 (R)

S, sensitive; R, resistant; I, intermediate.

Microbiological results

KPC-producing KPN isolates from patients 1 (ST307PT01), 2 (ST307PT02), 3 (ST307PT03), and 4 (ST307PT04) were resistant to all carbapenems, as well as to penicillin, levofloxacin, and cotrimoxazole. ST307PT01 was resistant to cefepime and gentamicin, while other isolates were susceptible to cefepime and gentamicin. ST307PT01 and ST307PT02 were susceptible to tigecycline, while ST307PT03 and ST307PT04 were resistant to tigecycline. Table 1 shows the MICs of the isolates. The susceptibility patterns generally corroborated the resistance genes identified through WGS (Table 2). Resistance plasmids were responsible for the majority of antimicrobial resistance genes; the only difference was that ST307PT01 has extra resistance genes, such as bla_CTX-M-15, aac(3)-IIa, aac(6′)Ib-cr, aacA4, bla_OXA-1, and catB4 due to the presence of a 10.9-kb region that is specific to pJYC01A plasmid. The other resistance genes identified were same in all isolates. Resistance genes associated with β-lactamases such as bla_TEM-1A, bla_SHV-28, bla_OXA-9, and bla_KPC-2 were identified in all isolates. Resistance genes associated with fluoroquinolone resistance, such as oqxA, oqxB, and QnrB66, were identified as well. The presence of bla_OXA-1 could explain the decreased susceptibility of ST307PT01 to cefepime.³⁷ The presence of aac(3)-IIa and aac(6′)Ib-cr could explain the decreased susceptibility of ST307PT01 to gentamicin.³⁸ The differences in phenotypic susceptibility to tigecycline and cefoxitin between isolates (ST307PT01 and ST307PT02 vs. ST307PT03 and ST307PT04) could not be explained by the differences in antimicrobial resistance genes identified by WGS. MLST corroborated these results, with all isolates belonging to the same sequence type (ST307). All isolates carried the bla_KPC-2 carbapenemase genes, which are contained within plasmidic Tn4401, a Tn3 family transposon.³⁹

Table 2.

Antimicrobial Resistance Genes Identified by Whole Genome Sequencing

Isolates	Aminoglycoside	β-Lactam	Fluoroquinolone	Fosfomycin	Tetracycline	Sulfonamide
ST307PT01	strA, strB, aac(3)-IIa(99.77%), aac(6′)Ib-cr	bla_KPC-2, blA_CTX-M-15, bla_OXA-1, bla_OXA-9 (99.88%), bla_TEM-1A (99.88%), bla_SHV-28	oqxA (98.89%), oqxB (98.57%), aac(6′)Ib-cr, QnrB66 (99.07%)	fosA (97.62%)	tet(A)	sul2
ST307PT02	strA, strB	bla_KPC-2_, bla_OXA-9 (99.88%), bla_TEM-1A (99.88%), bla_SHV-28	oqxA (98.89%), oqxB (98.57%), QnrB66 (99.07%)	fosA (97.62%)	tet(A)	sul2
ST307PT03	strA, strB	bla_KPC-2_, bla_OXA-9 (99.88%), bla_TEM-1A (99.88%), bla_SHV-28	oqxA (98.89%), oqxB (98.57%), QnrB66 (99.07%)	fosA (97.62%)	tet(A)	sul2
ST307PT04	strA, strB	bla_KPC-2, bla_OXA-9 (99.88%), bla_TEM-1A (99.88%), bla_SHV-28	oqxA (98.89%), oqxB (98.57%), QnrB66 (99.07%)	fosA (97.62%)	tet(A)	sul2

The genes were identified by ResFinder at a cutoff of 80% similarity to the database entries. The genes had 100% identity unless specified.

KPC, Klebsiella pneumoniae carbapenemase.

Genome sequence analysis

Complete genome sequences of the four clinical isolates were determined using the PacBio long read-based sequencing platform. Sequencing and assembly statistics are summarized in Supplementary Table S1. The circular structures of all chromosomes as well as the common ∼250-kb plasmids was confirmed using the Circlator tool. Their chromosomes were nearly identical in size (the length difference between the largest and smallest chromosomes was only 6,132 bp) and in %GC (57.44%). Mauve alignment showed a very similar organization between the isolate group and the references (Supplementary Fig. S1). Core gene alignment-based phylogenetic analysis indicated that all isolates and the reference ST307 strain were tightly clustered in a single group (Fig. 2). Average nucleotide identities between the four isolates were all ≥99.988%. SNP analyses showed that pairwise SNP distance between isolates were only 1–3, with the exception of ST307PT04 (46–49 SNPs, Supplementary Table S2), which suggests only 3 isolates among 4 KPC-producing KPN could have been transmitted from a common origin. To check where the chromosome length differences between strains come from, we compared each chromosome in all pairwise manner (Supplementary Table S3). Two through four near-identical, huge alignment blocks, separated by transposase genes (within insertion sequences) or other sequences at the equivalent sites, were identified. The largest spacing (4.2 kb) was considered to be the result of repeat-mediated deletion occurring only in ST307PT02, which makes it the smallest chromosome among the four strains. These observations clearly show that chromosome length differences could have caused from recent IS transposition and repeat-mediated deletion after these isolates diverged from a common origin. Their occupations at the equivalent sites, not random location, across all strains also support this hypothesis.

FIG. 2.

Core gene-based phylogenetic relationship estimated by maximum likelihood clustering of Klebsiella pneumoniae isolates with other references. Core gene calculation (≥99% identity across all genomes) and codon-based alignment performed using the Roary tool, which yielded a 3,431,253-bp codon-aware alignment out of 3,655 core genes. The approximately maximum likelihood phylogenetic tree was inferred using the FastTree 2 using –nt –gtr parameters. Strains sequenced in this study are indicated in bold. Numbers shown at the branch denote Shimodaira–Hasegawa local support values. Right panels show gene presence and absence patterns across 15 strains that were calculated using the Roary pipeline.

All KPN isolates harbored one or two plasmids. The common ∼250-kb resistance plasmids (pJYC01A, pJYC02A, pJYC03A, and pJYC04A), which belonged to the IncFII_K group with an additional FIB replicon,⁴⁰ were nearly identical in terms of nucleotide sequence and organization (Supplementary Fig. S2). As shown in Supplementary Fig. S3, this conjugative resistance plasmid has bla_KPC-2 contained within the Tn4401 “a” isoform.⁴¹ It is also notable that the resistance plasmid also harbors gene clusters for extra functions such as heavy metal resistance (copper, mercury, and arsenic), urea uptake, and type I restriction–modification system, which were also described from other KPN-derived plasmid pKPC_UVA02.⁴² pJYC01A was larger than the others due to a specific region within the 138,913–149,842 bp position (Supplementary Fig. S2). This segment, rich in additional antimicrobial resistance genes, appears to be deleted through a crossover between two identical IS6-family insertion sequences in the other resistance plasmids, pJYC02A, pJYC03A, and pJYC04A. The other resistance gene-rich region, on the opposite side of Tn4401a, was compared with other two similar plasmids, p72_FIBkpn (CP034282.1; 193,222 bp) and pRJ119-NMD1 (KX636095.1; 335,317 bp). The former had the highest blast score among all available 1,140 KPN plasmid sequences,^‡ while the latter was chosen due to the highest similarity in the multidrug resistance region (MRR) (Supplementary Fig. S4). Although they share common regions, extensive rearrangements are evident within MRR regions probably due to the recombination between insertion sequences.

ST307PT03 and ST307PT04 also have a common ColRNAI group plasmid, pJYC03B and pJYC04B, respectively. They do not have any antimicrobial resistance genes, except for genes encoding cloacin and its immunity protein.⁴³

Discussion

The spread of carbapenemase-producing KPN worldwide is considered a serious threat to health care facilities. To date, the most frequent class A carbapenemases are KPC enzymes, which is our focus herein.⁵ In the past decade, the outbreak of KPC-producing KPN was mainly caused by isolates belonging to clonal group 258.^44,45 Recently, however, new extensively drug resistant lineages have appeared globally. Among them, ST307 has been considered one of the most clinically relevant clones in various countries over the last 5 years.^46–49 The first outbreak of ST307 occurred in 2008 in Palermo, Sicily, Italy.⁵⁰ Patients infected with KPC-Kp ST307 had higher mortality rates (>50%) and longer hospital stays than those infected with other clones, indicating that this variant encodes more virulent factors.³⁵

KPC-producing KPN is known to be associated with the hospital environment; however, the frequency of community-acquired carbapenem-resistant Enterobacteriaceae (CRE) is expected to increase. In our experience, patient 1 (the index case) had no recent health-care facility exposure. There is only one reported case of a complicated urinary tract infection caused by KPC-producing KPN in a pregnant woman who had not been recently exposed to health care facilities.¹⁰ Although not limited to CPE, a study conducted in Taiwan reported that 23 of 89 patients with CRE (29.5%) were from community settings.⁵¹ Kelly et al. reviewed 10 studies and reported that 0.04–29.5% of cases were community-associated or community-initiated CRE.⁵² Their findings corroborate the emergence of potential CRE within communities and raise concerns regarding the introduction of new strains with abnormal clinical features. Pano-Pardo et al. reported that 15% of bloodstream infections caused by CPE were from community settings.⁵³ In their study, the majority of patients from community settings were immunocompromised owing to being afflicted with lymphoma, diabetes mellitus, cancer, or chronic kidney disease. Conversely, our study suggests that CPE has been identified in patients with no previous history of immunodeficiency.

Our epidemiologic survey of the infection control unit revealed that patients 1, 2, 3, and 4 had a history of contact in the ICU or one of the wards. Several studies have performed cultures of environmental surfaces and equipments to identify the transmission chain,^54–56 and discovered KPC-producing KPN on bed rails, mattresses, pumps, intravenous medication connectors, television monitors, and endotracheal tubes. However, this study was limited in that the environmental cultures were not included in our investigation. In our study, all isolates contained bla_KPC-2 genes, which are more likely to spread since their location in mobile genetic elements, such as plamids, make them move freely between Enterobacteriaceae. Usually, bla_KPC plasmids are known to be associated with ST258, but were found in ST307 in our study. The genetic structures surrounding bla_KPC-2 genes are contained within a Tn3-based transposon, Tn4401. Tn4401 was inserted at different loci and on plasmids varying in size and incompatibility group. Cuzon et al. demonstrated that Tn4401 is capable of transposition with 5-bp target site duplication and without target site specificity.³⁹ This work meant that KPC genes benefit from all the genetic tools available—active transposon, self-transferable plasmids, and efficient ST types—for their rapid spread to various bacterial species. Although the conventional investigation with epidemiological tracing and PFGE analysis suggested all four isolates could be clonally transmitted, WGS showed that only three isolates among four KPC-producing KPN could have been transmitted from a common origin. This shows WGS can help us to accurately characterize outbreaks as previous reports.⁵⁷

It is very difficult to control infection after the spread of carbapenemase-producing strains, and therapeutic agents are of limited efficacy. Therefore, the early detection of carbapenemase-producing strains, either as infections or colonies, is important to prevent the development of difficult or impossible-to-treat infections.³ Therefore, the phylogenetic tools applied to bacterial whole genome level is important in accurate epidemiological monitoring of multidrug-resistant pathogen outbreak.

Our experience of a hospital outbreak of KPC-2-producing KPN with the conjugative resistance plasmid linked with an index case of community-acquired infection suggests this resistant pathogen may spread not only in hospitals but also in community.

Our study has several limitations. Most importantly, SMRT using a PacBio RSII instrument can make the sequencing errors, and it may cause inaccuracy of sequencing alignment. We could not perform Illumina sequencing in this study, and this is an insurmountable limitation. However, the main purpose of this study is to introduce an outbreak of KPC-producing KPN linked with an index case of community-acquired KPC-producing isolate, and to investigate the molecular characteristics of the isolates, not a very precise genome analysis, so the shortcomings of PacBio SMRT sequencing might not be very critical weaknesses in this study. The errors in the final assembly obtained by PacBio SMRT sequencing may not have a critical impact on the results of this study, such as the detection of antibiotic-resistant genes and the analysis of MLST.

Conclusions

This report suggests that KPC-2-producing KPN with the conjugative resistance plasmid may increasingly spread not only in hospitals but also in community, and WGS can help to accurately characterize outbreaks. Unfortunately, it is likely that clinicians will face such a resistant pathogen in hospital outbreaks and community-acquired infections.

Footnotes

Disclosure Statement

No competing financial interests exist.

Supplementary Material

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