Emergence of Carbapenem-Resistant Serotype K1 Hypervirulent Klebsiella pneumoniae Strains in China

Abstract

We report the emergence of five carbapenem-resistant K1 hypervirulent Klebsiella pneumoniae (hvKP) strains which caused fatal infections in hospital patients in Zhejiang Province, China, upon entry through surgical wounds. Genotyping results revealed the existence of three genetically related strains which exhibited a new sequence type, ST1797, and revealed that all strains harbored the magA and wcaG virulence genes and a plasmid-borne bla_KPC-2 gene. These findings indicate that K1 hvKP is simultaneously hypervirulent, multidrug resistant, and transmissible.

TEXT

Hypervirulent Klebsiella pneumoniae (hvKP) is known to cause life-threatening infections such as liver abscess and necrotizing fasciitis (1). Characterized by the ability to produce the hypermucoviscous phenotype (2), hvKP was first reported in China in 1982, followed by sporadic emergence in various countries (1, 3). Importantly, young and relatively healthy individuals are often prone to hvKP infections, with diabetes being a major risk factor (4). K1 and K2 hvKP are the most clinically important serotypes due to their strong antiphagocytic ability and serum resistance (3, 5); interestingly, hvKP is rarely resistant to antibiotics. Except for an intrinsic resistance to ampicillin, most strains are susceptible to commonly used drugs (6). Due to the dissemination of mobile genetic elements encoding the KPC, NDM, and OXA-48 types of carbapenemase (7), however, antibiotic-resistant hvKP has begun to emerge in the past few years (8–10), posing a threat to human health. This study reports the emergence of carbapenem-resistant K1 hvKP strains in China, with a focus on their genetic and epidemiological features.

A retrospective study was conducted on 148 meropenem-resistant K. pneumoniae strains collected from two hospitals in Zhejiang Province in 2013, each being isolated from a single patient. Among these isolates, five K1, one K2, and five non-K1/K2 hvKP strains were identified by the loop string test (2) and confirmed by serotype-specific PCR, representing an isolation rate of 7.4%. A carbapenem-resistant K1 hvKP strain, KP70-2, was isolated from sputum and blood samples of a 50-year-old patient in December 2013; a carbapenem-susceptible K1 hvKP strain, KP70-1, was isolated from a sputum sample of the same patient a week before, when he was admitted for operative treatment of brain injury. The patient, who received imipenem for 6 days prior to isolation of KP70-2, was then treated with cefoperazone-sulbactam but died of septic shock 4 days later (Table 1). The strains KP70-1 and KP70-2, both belonging to ST23, shared similar but not identical pulsed-field gel electrophoresis (PFGE) patterns (Fig. 1). PCR screening of carbapenemase genes showed that KP70-2 carried the bla_KPC-2 element, whereas KP70-1 was neither resistant to carbapenem nor bla_KPC-2 positive (Table 2). S1-PFGE and Southern hybridization studies showed that both strains contained an ∼200-kb plasmid, yet bla_KPC was detectable only in the plasmid in KP70-2 (Fig. 2). Conjugation experiments, performed as previously described (11), showed that this bla_KPC-carrying plasmid was not transferable to Escherichia coli. Importantly, the magA, rmpA, and wcaG virulence genes were detectable in both strains, among which rmpA was found located in the ∼200-kb plasmid.

TABLE 1.

Characteristics of KPC-2-producing K1 hvKP strains and clinical information for infections that they caused^a

Strain identifier	Isolation date (day/mo/yr)	Gender/age (yr)	Isolation site(s)	Underlying disease or condition	Treatment	Outcome	Serotypes	MLST type
KP70-1	30/11/2013	M/50	Sputum	Brain injury	IMI		K1	ST23
KP70-2	6/12/2013		Sputum, blood		SBT-CPZ	Septic shock, death	K1	ST23
KP1088-1	2/10/2013	M/67	Sputum	Multiple injuries	IMI		K1	ST23
KP1088-2	12/10/2013		Sputum, blood		LNZ	Septic shock, death	K1	ST23
KP86	23/4/2013	M/88	Sputum, blood	Abdominal infection	TZP, TEIC, VA, SBT-CPZ, FLC	Septic shock, death	K1	ST1797
KP91	5/5/2013	F/73	Sputum, blood	Septic arthritis	TZP, LNZ	Septic shock, death	K1	ST1797
KP94	6/5/2013	M/32	Sputum	Finger injury	TZP, TEIC, ICZ	Septic shock, death	K1	ST1797

^aAbbreviations: M, male; F, female; IMI, imipenem; SBT-CPZ, sulbactam-cefoperazone; LNZ, linezolid; TZP, piperacillin-tazobactam; FLC, fluconazole; TEIC, teicoplanin; VA, vancomycin; ICZ, itraconazole. Multilocus sequence typing (MLST) was performed according to the protocol described on the K. pneumoniae MLST website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html).

FIG 1.

PFGE patterns of K1 hvKP strains. KP1088-1 and KP1088-2, isolated from the same patient, exhibited varied PFGE patterns; the same phenomenon was observable in strains KP70-1 and KP70-2, which were isolated from a second patient. Strains KP86, KP91, and KP94, isolated from three patients in an intensive care unit, respectively, were genetically identical. The dendrogram depicting the genetic relatedness of the test strains was generated from the homology matrix with an 0.2% coefficient by the unweighted pair group method using arithmetic averages (UPGMA) to reveal the degree of relatedness between different PFGE profiles. Isolates were allocated to the same PFGE group if their dice similarity index was ≥85%.

TABLE 2.

MICs and carriage of virulence and carbapenemase genes in hvKP and selected transconjugants

Strain identifier	MIC (mg/liter) of druga:												Virulence genes	Carbapenemase gene
	CAZ	CTX	CRO	CFP-SUL	IMP	MEP	AMK	TIC-CLA	GEM	CIP	CL	TGC
KP70-1	<0.5	<0.25	0.064	0.25	0.25	0.032	2	4	0.5	0.032	<1	<1	magA, rmpA, wcaG
KP70-2	<0.5	16	128	64	32	32	1	>256	0.25	0.032	2	<1	magA, rmpA, wcaG	blaKPC-2
KP1088-1	<0.5	<0.25	0.064	0.25	0.25	0.032	2	4	1	0.032	2	<1	magA, rmpA, wcaG
KP1088-2	>32	>16	>256	64	>32	>32	>256	>256	>256	0.032	2	<1	magA, rmpA, wcaG	blaKPC-2
T-KP1088-2b	>32	>16	>256	32	2	4	>256	>256	>256	0.125	<1	<1		blaKPC-2
KP86	>32	>16	128	128	>32	>32	8	>256	2	8	<1	<1	magA, wcaG	blaKPC-2
KP91	>32	>16	128	128	>32	>32	8	>256	2	8	<1	<1	magA, wcaG	blaKPC-2
KP94	>32	>16	>256	128	>32	>32	8	>256	2	16	<1	<1	magA, wcaG	blaKPC-2

^aAbbreviations: CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; CFP-SUL, cefoperazone-sulbactam; IMP, imipenem; MEP, meropenem; AMK, amikacin; TIC-CLA, ticarcillin-clavulanic acid; GEM, gentamicin; CIP, ciprofloxacin; CL, colistin; TGC, tigecycline. The MICs were determined using the agar dilution method, and results were interpreted according to the CLSI criteria of 2015 (13). The 2014 EUCAST breakpoints were used (available at http://www.eucast.org/clinical_breakpoints/) for tigecycline.

^bT, transconjugant.

FIG 2.

KPC-2-encoding plasmids in hvKP. S1-PFGE and Southern hybridization with a bla_KPC-2 probe were performed for seven K1 hvKP isolates, including five carbapenem-resistant hvKP strains. The plasmid band(s) of strains KP1088-2, KP70-2, and KP94 in which the bla_KPC-2 gene was detectable was marked by an asterisk, a number sign, and arrows, respectively.

In the second case, a pair of carbapenem-susceptible and -resistant K1 hvKP strains was recoverable from a 67-year-old patient after surgical treatment of multiple injuries. These two strains, namely, KP1088-1 and KP1088-2, also belonged to ST23 type K1 hvKP and exhibited slightly different PFGE patterns (Table 1; Fig. 1). The first strain, KP1088-1, was isolated from a sputum sample of the patient, who then received imipenem treatment for 7 days prior to isolation of KP1088-2 from his sputum and blood samples. Strain KP1088-02 was resistant to carbapenems, presumably due to acquisition of a bla_KPC-2 gene during treatment, whereas the earlier strain, KP1088-1, was susceptible to carbapenems. The patient was then subjected to linezolid treatment but soon died of septic shock. S1-PFGE showed that strain KP1088-2 had acquired two additional ∼75-kb and ∼100-kb plasmids compared to KP1088-1. Southern hybridization indicated that the bla_KPC-2 gene was located on the ∼100-kb plasmid (Fig. 2), which was transferable to E. coli by conjugation. Both strains were found to harbor the magA, rmpA, and wcaG genes, with rmpA detectable in an ∼200-kb virulence plasmid (Table 2).

The third incident involved isolation of three carbapenem-resistant K1 hvKP strains from separate patients in an intensive care unit (ICU) within a 2-week period (Table 1). This was considered a small outbreak since the three isolates exhibited almost identical PFGE patterns (Fig. 1). Two of the strains were isolated from sputum and blood samples of two elderly patients suffering from intra-abdominal infection and septic arthritis, respectively. The last case involved a 32-year-old patient, originally a healthy young individual without major underlying diseases, with finger injury who required a surgical operation. Strain KP94 was isolated from the sputum sample of this patient upon admission into ICU. All three isolates were found to belong to a new sequence type, ST1797; harbored the magA and wcaG virulence genes but not the rmpA-bearing virulence plasmid, and were resistant to most antibiotics tested (Table 2). S1-PFGE hybridization indicated that, in each case, bla_KPC-2 was detectable in two plasmids with sizes of ∼50 kb and ∼600 kb (Fig. 2), which were not transferable to E. coli.

In contrast to the fatal outcomes of K1 hvKP infections, all patients infected by carbapenem-resistant K2 and non-K1/K2 hvKP strains fully recovered after treatment with carbapenems or other antibiotic combinations. Compared to the mortality rate of liver abscess caused by hvKP (2 to 18%), all five cases of KPC-2-producing K1 hvKP infection described in this work had fatal outcomes regardless of the health status of the patients (1, 12). In addition, such infections were associated with trauma, abdominal infection, and purulent arthritis, respectively, which are rarely linked to hvKP infections. The invasiveness of these strains was reflected by the observation that organisms were often recoverable from both blood and sputum specimens even though the site of infection was apparently a surgical wound. Acquisition of carbapenem resistance by K1 hvKP was likely induced by imipenem treatment, suggesting that these strains are capable of acquiring a plasmid harboring the bla_KPC-2 gene, or by incorporating a mobile bla_KPC-2-bearing DNA fragment, likely Tn4401 in this case, into the virulence plasmid that already exists in the K1 hvKP host. These characteristics of K1 hvKP infection imply that K1 hvKP is simultaneously hypervirulent, multidrug resistant, and transmissible in the hospital setting.