Two Hypervirulent Klebsiella pneumoniae Isolates Producing a bla_KPC-2 Carbapenemase from a Canadian Patient

This report describes two hypervirulent Klebsiella pneumoniae isolates that produced K. pneumoniae carbapenemase (KPC), which were identified from a rectal swab and a urine culture upon hospital admission. The patient had recently traveled to Greece, where he was hospitalized.

ABSTRACT

This report describes two hypervirulent Klebsiella pneumoniae isolates that produced K. pneumoniae carbapenemase (KPC), which were identified from a rectal swab and a urine culture upon hospital admission. The patient had recently traveled to Greece, where he was hospitalized. The isolates were sequence type 86 and contained an IncHI1B IncFIB_K hypervirulent plasmid and an IncFII_K plasmid harboring KPC.

TEXT

Hypervirulent Klebsiella pneumoniae (hvKp) was first identified in Taiwan in 1986 (1) and since has spread throughout the Asia Pacific region, with increasing numbers of cases being reported in Europe and the Americas (2). Although rare in Canada, hvKp strains have been reported in community-acquired bacteremia and have been associated with the K2 serotype and the presence of rmpA (3).

In contrast to classic K. pneumoniae, hvKp is characterized by its acquisition of a cluster of virulence factors, which can be encoded on a virulence plasmid as well as within the chromosome (2). Clinically, hvKp is associated with an ability to cause life-threatening infections in healthy individuals from the community (2). hvKp strains frequently belong to capsular serotype K1 or K2 and commonly harbor a regulator of mucoid phenotype A (rmpA) and iron acquisition regulatory genes (iuc-type and iro-type) (4).

Antibiotic resistance in hvKp is rare; however, reported cases are increasing worldwide and represent a major public health concern (2). Specifically, carbapenem resistance in hvKp strains due to the presence of carbapenemase genes such as bla_KPC (5–7), bla_NDM (8), and bla_OXA-232 (9) on mobile plasmids has been observed in China and the United Kingdom. Here we report two hvKp isolates harboring the known virulence plasmid pLVPK in addition to plasmid-mediated K. pneumoniae carbapenemase-2 (KPC-2).

A male patient >90 years of age was admitted to a hospital in Montreal, Canada, in September 2018, complaining of progressive confusion and difficulty walking. These symptoms had been progressing for approximately 2 months. A computed tomography (CT) scan of the head was performed and revealed a subdural hematoma that required craniotomy and hematoma evacuation. Upon questioning, it was found that the patient had sustained a head injury 2 months earlier while visiting Greece and required a short stay in an acute care hospital. Care during that stay included a CT scan of the head and laceration closure with stitches.

Given the presence of low-level nosocomial transmission of carbapenemase-producing Enterobacteriaceae (CPE) at the Canadian hospital (2018 institutional nosocomial infection acquisition rate, 1.2 cases per 10,000 patient-days; Yves Longtin, unpublished data), upon admission the patient was screened for CPE carriage according to the local infection control policy, using rectal swab sampling and a chromogenic plate (Carbasmart; bioMérieux, Saint-Laurent, Canada). Suspected colonies were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Vitek system; bioMérieux), tested for the presence of a carbapenemase gene by PCR (10), and tested for hypermucoviscosity/hypervirulence with a string test. The carbapenemase gene bla_KPC was identified in a string-test-positive K. pneumoniae isolate (isolate 11A18CPO60). Additionally, the patient presented 6 days later with a urinary tract infection due to a carbapenem-resistant, string-test-positive, KPC-producing K. pneumoniae isolate (isolate N19-00083), which was treated with trimethoprim-sulfamethoxazole for 14 days. The patient was discharged in October 2018. Extensive screening of contacts did not identify nosocomial transmission.

Phenotypic antimicrobial susceptibility testing was conducted for both isolates (Sensititre; Trek Diagnostics, Oakwood Village, OH, USA) (11) (Table 1). The isolates were sent for whole-genome sequencing (WGS) using NextSeq (Illumina, San Diego, CA, USA) and MinION (Oxford Nanopore Technologies, Oxford, UK) systems. A complete chromosome 18CPO060 and two circularized plasmids, i.e., a hypervirulent plasmid (phvKP060 [218 kb]) and a KPC-2 plasmid (pKPCKP060 [109 kb]), were resolved from the primary isolate and deposited in GenBank.

TABLE 1.

Susceptibility profiles of two hvKp isolates harboring bla_KPC-2

Antimicrobial	Isolate 11A18CPO060		Isolate N19-00083
	MIC (mg/liter)	Susceptibilitya	MIC (mg/liter)	Susceptibility
Amikacin	≤4	S	≤4	S
Aztreonam	16	R	16	R
Cefepime	≤2	S	4	I
Cefotaxime	4	R	8	R
Ceftazidime	8	I	8	I
Ciprofloxacin	≤0.25	S	≤0.25	S
Colistin	≤0.25	S	≤0.25	S
Doripenem	0.5	S	1	S
Doxycycline	≤2	S	≤2	S
Ertapenem	2	R	1	I
Gentamicin	≤1	S	≤1	S
Imipenem	4	R	2	I
Levofloxacin	≤1	S	≤1	S
Meropenem	≤1	S	≤1	S
Minocycline	≤2	S	≤2	S
Piperacillin-tazobactam	>64/4	R	>64/4	R
Polymyxin B	≤0.25	S	≤0.25	S
Ticarcillin-clavulanic acid	>128/2	R	>128/2	R
Tigecycline	≤0.25	S	≤0.25	S
Tobramycin	≤1	S	≤1	S
Trimethoprim-sulfamethoxazole	≤0.5/9.5	S	≤0.5/9.5	S

^aS, susceptible; I, intermediate; R, resistant.

The 11A18CPO60 isolate belonged to serotype KL2 and contained the virulence genes rmpA, rmpA2, iroBCDN, iucABCD, iutA, ybtAEPQSTUX, fyuA, irp1, and irp2, as determined using a local installation of Kleborate (https://github.com/katholt/Kleborate). The isolate contained the β-lactamase and fosfomycin resistance genes bla_SHV-1 and fosA, respectively, in addition to KPC-2, and belonged to sequence type 86 (ST86) (Center for Genomic Epidemiology). Although rarely reported, hvKp ST86 has been associated with serotype K2 in China (4) and an extended-spectrum β-lactamase (ESBL)-producing hvKp strain in France (12). A study by Zhao et al. showed that K. pneumoniae ST86 had more virulence traits and elevated mouse lethality (mean 50% lethal dose [LD₅₀] of 1.7 × 10⁴ CFU/mouse), compared to other hvKp STs (13). Additionally, infections caused by K. pneumoniae ST86 have been associated with high mortality rates (41.6%), which are thought to be due to increased virulence, compared to other STs (14), suggesting the potential public health threat of this isolate. The hypervirulent plasmid (phvKP060) belonged to plasmid incompatibility groups HI1B and FIB_K and was closely related to the previously described virulence plasmid pLVPK (GenBank accession no. AY378100), which was mapped with 98% coverage using Plasmid Profiler (15). Similar to pLVPK, phvKP060 contained the regular capsular polysaccharide genes rmpA and rmpA2 along with the iron acquisition system iroBCDN, a salmochelin operon, and iucABCD-iutA, an aerobactin operon. The second plasmid from this isolate, pKPCKP060, belonged to incompatibility group FII_K, a well-described plasmid group associated with KPC (16). The closest match to pKPCKP060 in the NCBI database was pKpn70747 (GenBank accession no. CP023443) from an unpublished K. pneumoniae plasmid (89% coverage and 99% identity). The bla_KPC-2 gene was harbored on Tn4401a. The KPC-2 variant has been observed in only 2.1% of KPC cases (5/237 cases) at the Montreal hospital site since 2009 (Yves Longtin, unpublished data). KPC-producing K. pneumoniae strains are well described as being endemic in Greece; specifically, one surveillance study showed that 100% of KPC producers from Greece with KPC-2 on Tn4401a demonstrated associated FII_K plasmids (17). The genetic context of KPC mirrored that observed in Greece and was extremely rare at our hospital site, suggesting that pKPCKP060 likely was acquired abroad. pKPCKP060 did not contain any additional antimicrobial resistance genes or known virulence genes.

Analysis of the urine isolate (isolate N19-00083) revealed an ST86 isolate with the same resistance and virulence genes, as well as the hypervirulent plasmid and the KPC plasmid seen in the primary isolate (isolate 11A18CPO60). Unlike isolate 11A18CPO60, isolate N19-00083 also contained a 3,789-bp ColE plasmid. Single-nucleotide variant (SNV) typing was conducted (https://github.com/phac-nml/snvphyl-galaxy) using the total genetic content of isolate 11A18CPO60 as the reference. A total of 14 SNV differences between the two isolates, distributed throughout the core genome, were observed. Given the number of SNVs in the short hospital stay and the additional plasmid in the urine isolate, it is possible this patient was colonized by two different K. pneumoniae ST86 strains that were derived from a common ancestor.

To our knowledge, this is the first reported case of a hvKp strain harboring a carbapenemase in North America. Clinicians and laboratories should be aware of carbapenemase-producing hvKp strains in high-risk individuals. The presence of KPC-2 and the large number of virulence genes in this isolate are of concern.

Data availability.

The plasmids and chromosome have been deposited in GenBank under accession numbers CP034776 to CP034778.