Emergence of Klebsiella pneumoniae Coproducing KPC-2 and 16S rRNA Methylase ArmA in Poland

Abstract

A Klebsiella pneumoniae epidemic strain that coproduced carbapenemase KPC-2 (K. pneumoniae carbapenemase 2) and 16S rRNA methylase ArmA has emerged in Poland. Four nonduplicate isolates from patients in a hospital in Warsaw, Poland, were found to carry the bla_KPC-2 and armA genes on ca. 50-kb and 90-kb plasmids, respectively. Tn4401 with a 100-bp deletion in the variable region was detected in all the isolates. XbaI pulsed-field gel electrophoresis (PFGE) revealed 93.2% similarity of the isolates. All the isolates were resistant to carbapenems and 4,6-disubstituted 2-deoxystreptamines.

Klebsiella pneumoniae carbapenemase (KPC) is a molecular class A serine β-lactamase belonging to functional group 2f. This enzyme hydrolyzes β-lactams of several different classes, including carbapenems (18). KPC was first reported in the United States in 2001 (18, 25). This enzyme is encoded by the bla_KPC gene, which maps to transposon Tn4401 that has been reported to occur in a variety of transferable plasmids (12, 16). Despite a highly conserved amino acid sequence of KPC, several variants of the bla_KPC gene have been distinguished (http://www.lahey.org/Studies/other.asp#table1). KPC has recently become a major therapeutic challenge with the KPC-2 and KPC-3 variants predominating in the United States and Europe (14, 18, 19, 23). Besides clonal dissemination in hospitals (17), bla_KPC has also been found to spread laterally, even at the interspecies level (20). Enterobacter cloacae producing KPC-2 together with 16S rRNA methylase ArmA was recently reported in China (23).

Plasmid-encoded 16S rRNA methylases have emerged in members of the family Enterobacteriaceae as a new mechanism of resistance to 4,6-disubstituted 2-deoxystreptamines, which encompass a majority of clinically important aminoglycosides (24). In contrast to substrate-specific aminoglycoside-modifying enzymes, the methylases confer high-level resistance by alteration of the 16S rRNA A site. Among 16S rRNA methylases described thus far (5), ArmA is a global concern (7, 9, 15). This methylase was first detected in Citrobacter freundii in Poland (11) and currently predominates in Europe (2, 11). ArmA is encoded by the armA gene (8), which maps to composite transposon Tn1548 (9) located on a large (ca. 80- to 90-kb) conjugative plasmid that also carries a gene encoding a CTX-M-type extended-spectrum β-lactamase (ESBL) (11).

Herein, we report emergence of Klebsiella pneumoniae coproducing carbapenemase KPC-2 and 16S rRNA methylase ArmA in Poland. To the best of our knowledge, this is the first such report in Europe.

We tested five nonduplicate clinical isolates of K. pneumoniae collected from patients in a hospital in Warsaw, Poland. All five isolates were considered etiologic agents of infections, mostly urinary tract infections (see Fig. 1A for details). The isolates were identified using Vitek 2 (bioMerieux, France) and were found resistant to ertapenem and meropenem. Four isolates were also resistant to various aminoglycosides. Agar dilution and Etest strips (AB bioMerieux, Solna, Sweden) were used to determine susceptibility, and MICs were interpreted according to the CLSI recommendations (4). Phenotypic test with boronic acid (21) showed that all tested strains produce carbapenemase. High-level resistance to various 4,6-disubstituted 2-deoxystreptamines (MIC of 1,024 μg/ml) was detected in all but one (DM0267) of the tested isolates (Table 1.) This finding suggested 16S rRNA methylase activity. Consequently, all the tested isolates were examined by PCR (8, 12) for the bla_KPC and armA genes. The bla_KPC gene was detected in all isolates tested, while armA was found in four isolates (Table 1). The bla_KPC variant expressed by the isolates tested was determined by DNA sequencing with primers KPC895F (5′-TTGATGTCACTGTATCGCCG-3′) and KPC895R (5′-TTTCAGAGCCTTACTGCCCG-3′) (the forward and reverse primers are indicated by F and R at the end of the primer designation, respectively) using the BigDye terminator V.3.1. cycle sequencing kit (Applied Biosystems, Foster City, CA). The primers were designed for the reference bla_KPC-2 gene sequence (GenBank accession number AY034847). All strains tested were found to carry the bla_KPC-2 gene for KPC-2. This variant of KPC has been recently reported in Poland in K. pneumoniae ST258 (1). Therefore, we determined the multilocus sequence type of isolate DM0269 according to the protocol described on the K. pneumoniae multilocus sequence typing (MLST) website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). Our results showed that isolate DM0269 also belongs to the ST258 clone.

FIG. 1.

(A) Dendrogram of the tested K. pneumoniae isolates based on XbaI PFGE profiles. The XbaI PFGE pattern, isolate, presence (+) or absence (−) of the armA gene, ward, and date (sample collection date) are shown. URL, urological department; INT, internal diseases department; Tol, tolerance. (B) Plasmid profiles of K. pneumoniae isolates with bla_KPC and armA carriers delineated by the Southern blot DNA/DNA hybridization. Lanes 1 to 5 show the plasmid profiles of strains DM0267, DM0270, DM0265, DM0266, and DM0269, respectively. Lanes 6 and 7 show the hybridization results for the plasmid profile of isolate DM0269 and molecular probes specific for bla_KPC and armA, respectively. Chr, chromosomal DNA fragments. (C) Plasmids in E. coli DH5α transformants. ETbla_KPC and ETarmA, plasmids pETKp50 and pETKp90 from isolate DM0269, respectively. Lane W, plasmids from the wild donor. (D) PstI restriction endonuclease profiles of plasmids in E. coli DH5α transformants with bands to which bla_KPC and armA probes hybridized. PstI profiles of plasmids from ETbla_KPC (lane 1), ETarmA (lane 3), and E. coli Δ32/01 (lane 5) together with Southern blot hybridization with bla_KPC (lane 2) and armA (lanes 4 and 6) probes. Lane M, DNA ladder (λ phage HindIII digested). The approximate size of DNA is shown in kilobases to the left of the gels in panels B to D. The picture was electronically edited to show DNA electrophoretic patterns and the corresponding Southern blot hybridization results together.

TABLE 1.

Characterization of the tested strains

Strain	Presence or absence of resistance genea:			Susceptibility (MIC [μg/ml]) to the following antimicrobial agentb:
	armA	blaKPC	blaCTX-M	GEN	AMK	KAN	NEO	STR	IMP	MEM	ETP	CTX	CTX CLA	CAZ	TET	CIP	CST	TGC
K. pneumoniae
DM0265	+	+	+	1,024	1,024	1,024	256	16	16	>32	>32	32	32	128	4	64	0.5	1
DM0266	+	+	+	1,024	1,024	1,024	256	16	16	>32	>32	64	32	64	4	64	0.5	1.5
DM0267	−	+	−	2	512	64	256	16	128	>32	>32	64	32	64	4	64	0.5	1
DM0270	+	+	+	1,024	1,024	1,024	256	16	128	>32	>32	128	64	512	8	32	0.5	1.0
DM0269	+	+	+	1,024	1,024	1,024	512	16	128	>32	>32	128	64	256	4	64	0.5	1.5
E. coli
ETblaKPCc	−	+	−	0.125	0.25	1	0.5	0.125	1	0.5	2	4	0.038	16	1.50	0.05	0.094	0.38
ETarmAd	+	−	+	128	256	512	1	4	0.19	0.023	0.008	>32	0.016	1.50	1.50	0.032	0.094	0.38
DH5α	−	−	−	0.125	0.25	1	0.5	0.125	0.125	0.016	0.004	0.023	0.016	0.25	1	0.023	0.094	0.38

^aThe presence (+) or absence (−) of resistance genes armA, bla_KPC, and bla_CTX-M was determined by the PCR assay.

^bGEN, gentamicin; AMK, amikacin; KAN, kanamycin; NEO, neomycin; STR, streptomycin; IMP, imipenem; MEM, meropenem; ETP, ertapenem; CTX, cefotaxime; CTX CLA, cefotaxime with clavulanic acid (2.0 μg/ml); CAZ, ceftazidime; TET, tetracycline; CIP, ciprofloxacin; CST, colistin; TGC, tigecycline.

^cE. coli DH5α transformed by 50-kb plasmid from isolate DM0269.

^dE. coli DH5α transformed by 90-kb plasmid from isolate DM0269.

Plasmids were extracted from the tested strains using the NucleoBond PC 20 kit (Macherey-Nagel, Germany). Four tested strains harbored three large plasmids (Fig. 1B) with estimated sizes of 50, 90, and 180 kb. Isolate DM0267, which was armA negative, lacked the 90-kb plasmid. Southern blot analysis was performed as previously described (22) to determine which plasmids carry the bla_KPC-2 and armA genes. The probes specific for these two genes hybridized with 50-kb and 90-kb plasmids, respectively (Fig. 1B). Multiple electroporation experiments were carried out to transform Escherichia coli DH5α with the 50- and 90-kb plasmids from K. pneumoniae DM0269 by the method of Kitchel et al. (14). Kanamycin (64 μg/ml) and amikacin (64 μg/ml) were used to select transformants producing ArmA. Transformants ETbla_KPC and ETarmA, which were resistant to carbapenems and 4,6-disubstituted 2-deoxystreptamines (Table 1), carried plasmids pETKp50 (50 kb) and pETKp90 (90 kb), respectively (Fig. 1C). The armA gene carrier (pETKp90) belonged to the L/M incompatibility group as determined by PCR typing with primers described by Galimand et al. (9). Moreover, PstI restriction endonuclease analysis followed by Southern blot hybridization with the armA probe showed that pETKp90 is highly related to the pCTX-M3-type plasmid of E. coli Δ32/01 (Fig. 1D). This plasmid originated from CTX-M-3-producing clinical isolate 32/01 of Salmonella enterica serovar Oranienburg, which was isolated in Poland in 2001 (10). In contrast, the replicon type of pETKp50 encoding KPC-2 could not be determined by the aforementioned primers and primers described by Carattoli et al. (3). The PstI pattern of pETKp50 was dissimilar to that of pETkp90 (Fig. 1D).

Other studies have shown that the bla_KPC-2 gene occurs in a variety of plasmids ranging from 24 to 120 kb (1, 12). Nevertheless, it was most frequently reported in plasmids from 50 kb to 75 kb (14, 18), which is in concordance with our results. In contrast, human clinical isolates producing ArmA were reported to bear the armA gene on a conjugative plasmid of ca. 90 kb that was similar to pCTX-M3 (11). Because pCTX-M3 carries a gene encoding CTX-M-3 ESBL, we carried out PCR assay with primers specific for the bla_CTX-M gene family (6). All the isolates tested except for DM0267 were found to produce CTX-M-type ESBL (Table 1). Since pCTX-M3 and its derivatives were commonly detected in Enterobacteriaceae in Poland, it may explain the presence of the pCTX-M3-type plasmids in the isolates tested (2, 9, 11). In contrast, the origin of the plasmid carrying the gene encoding KPC-2 remains unclear. The only KPC-producing K. pneumoniae isolates reported so far in Poland carried the bla_KPC-2 gene on a large plasmid that was 110 kb in size (1). The isolates tested, however, bear this gene on the 50-kb plasmid. To better characterize this plasmid, PCR assay with primers specific for nonconserved region of the Tn4401 was carried out (14). All the isolates tested yielded a ca. 604-bp amplicon corresponding to a Tn4401 variant with a 100-bp deletion (data not shown). Interestingly, this variant is relatively rare. It was found in K. pneumoniae ST258 in the United States and Israel (14). The results of the transposon analysis suggested that the isolates tested were clonal. Pulsed-field gel electrophoresis (PFGE) was therefore conducted (14). PFGE patterns were analyzed using GelCompar II version 5.10 software (Applied Maths, Sint-Martens-Latem, Belgium). Four genotypes were distinguished (Fig. 1). The very high similarity (93.5%) of the genotypes suggested that all the isolates tested belong to the same strain sensu stricto.

In conclusion, our results demonstrate that K. pneumoniae in Poland has accumulated two broad-spectrum resistance traits: KPC carbapenemase and 16S rRNA methylase ArmA. Due to its resistance to ciprofloxacin, cephalosporins, carbapenems, and a spectrum of aminoglycosides, this strain disseminated among patients in two hospital wards and became a real challenge for the hospital authorities. Our findings suggest that coproduction of KPC-2 and ArmA is a novel strategy developed by K. pneumoniae to survive in hospitals if aminoglycosides and carbapenems are administrated in combination. This hypothesis is also supported by Jiang and coworkers (13), who found a clinical isolate of K. pneumoniae that carried bla_KPC-2 and armA in a large multidrug resistance plasmid in China. Moreover, strong selective pressure favoring coproduction of KPC-2 and ArmA by hospital strains was also observed in a clinical isolate of Enterobacter cloacae in China (23). This isolate carried bla_KPC-2 and armA on two large plasmids, which resembles the phenomena reported herein for K. pneumoniae.