Rapid Expansion of KPC-2-Producing Klebsiella pneumoniae Isolates in Two Texas Hospitals due to Clonal Spread of ST258 and ST307 Lineages

Abstract

During 2007 to 2010, 23 carbapenem-resistant Enterobacteriaceae strains were recovered in two hospitals in Texas among three institutions surveyed. Eighteen (2.0% overall) strains carried bla_KPC-2 and were collected in 2008 (3/150; 2.0%) and 2010 (15/289; 5.1%). Clonality was noted within hospitals and ten isolates belonged to ST258. All strains carried bla_KPC-2 on plasmids and 14 had this gene embedded in Tn4401a. Tn3 transposase was detected upstream of bla_KPC-2 in two isolates and another strain had two adjacent copies of bla_KPC. Surveillance in prior years shows sporadic detection of KPC-producers in Texas, however, a recent increase in carbapenem-resistant Enterobacteriaceae in this state was due to the spread of KPC-producing Klebsiella pneumoniae, including isolates from ST258.

B la _KPC was first detected in a Klebsiella pneumoniae clinical isolate collected in 1996 from a patient hospitalized in North Carolina,⁸ and KPC variants remain the most clinically significant β-lactamase among Class A derivatives. KPC-producing Enterobacteriaceae have been reported in 36 USA states, Washington DC, and Puerto Rico³ and although several outbreaks of isolates producing this serine-carbapenemase have been reported in the Northeast region of the USA, where these strains become endemic,⁶ this gene is still more rarely detected in other USA regions.

Overall, the dissemination of bla_KPC has been associated with clonally related strains. Moreover, there is mounting evidence that bla_KPC genes are also consistently associated with a specific genetic element (i.e., transposon Tn4401).⁵ Tn4401 is a Tn3-like transposon that has been identified in distinct plasmids carried by KPC-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates from various geographic areas.² In addition, the majority of bla_KPC-2-carrying K. pneumoniae belong to the multilocus sequence type (MLST) 258 and double-locus variants, which may have contributed to the worldwide dissemination of KPC-producing strains.^3,7

KPC-producing strains have been recently reported in a tertiary care hospital in Houston, Texas,⁴ but little is known about the genetic characteristics and dissemination of these isolates. In this study, we report a recent increase in the prevalence of carbapenem-resistant Enterobacteriaceae in Texas hospitals participating in the SENTRY Antimicrobial Surveillance Program and provide the molecular epidemiology characteristics of these isolates.

A total of 879 Enterobacteriaceae clinical isolates were submitted from three hospitals in Houston, Texas area (372, 177, and 330 from hospital A [Houston], B [Houston], and C [Galveston], respectively), as part of the SENTRY Program (2007–2010), and were susceptibility tested according to the Clinical and Laboratory Standards Institute (CLSI) recommendations.¹ Among these strains, 23 (2.6% of total) isolates collected from hospitals A and B (both in Houston) displayed MIC results for imipenem or meropenem of≥2 μg/ml and were selected for further analysis (Table 1). These strains belonged to four bacterial species as follows: K. pneumoniae (20 strains), Serratia marcescens (one strain), Enterobacter aerogenes (one strain), and Citrobacter freundii (one strain). All 23 selected strains were recovered from blood cultures and displayed elevated MIC results for the majority of antimicrobial agents tested, except for tigecycline (0.12 to 2 μg/ml). Seven of 20 (35.0%) K. pneumoniae were also resistant to colistin (Table 1).

Table 1.

Carbapenem-Resistant Enterobacteriaceae Clinical Isolates Collected in Two Medical Institutions in Texas (2007–2010)

			MIC (μg/ml)
Hospital	Organism (no.)	Year	IPM	MER	P/T	AZT	CAZ	CRO	CPM	AMK	GEN	CIP	TET	TGC	COL	Age	Sex	Culture Date	PFGE	ST
KPC-2-producing strains (18)
A	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	0.5	>8	>4	>8	2	1	83	F	6-Jun	B1
A	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	1	>8	>4	8	1	≤0.5	50	M	19-Aug	B4
A	K. pneumoniae	2010	8	>8	>64	>16	32	>32	16	1	≤1	>4	>8	1	≤0.5	NA	NA	NA	D	494
B	C. freundii	2008	4	4	>64	>16	16	32	8	0.5	>8	4	4	1	≤0.5	81	M	18-May
B	K. pneumoniae	2008	>8	8	>64	>16	>32	>32	>16	32	≤1	>4	8	1	>4	68	F	5-Oct	A1
B	K. pneumoniae	2008	>8	8	>64	>16	>32	>32	16	32	≤1	>4	4	1	>4	68	F	25-Oct	A1
B	K. pneumoniae	2010	8	>8	>64	>16	>32	>32	>16	32	≤1	>4	4	0.5	≤0.5	57	M	7-Jan	B3	307
B	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	32	≤1	>4	4	0.5	>4	51	M	17-Mar	A1
B	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	32	2	>4	4	0.5	≤0.5	57	F	18-May	E	258
B	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	16	≤1	>4	4	0.5	≤0.5	57	F	22-May	E
B	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	32	2	>4	4	1	>4	72	M	29-May	A1	258
B	K. pneumoniae	2010	8	>8	>64	>16	>32	>32	>16	32	2	>4	4	0.5	>4	72	M	8-Jun	A1
B	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	0.5	8	>4	>8	0.5	≤0.5	58	M	17-Jul	B1	307
B	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	32	>8	>4	>8	0.25	≤0.5	59	F	12-Oct	B2
B	K. pneumoniae	2010	8	8	>64	>16	8	>32	4	1	≤1	≤0.03	2	0.25	>4	63	F	10-Nov	C	901
B	K. pneumoniae	2010	8	8	>64	>16	>32	>32	16	32	≤1	>4	4	0.5	≤0.5	61	F	3-Dec	A3
B	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	>32	2	>4	4	1	≤0.5	58	F	3-Dec	A1
B	K. pneumoniae	2010	>8	>8	>64	>16	>32	>32	>16	32	2	>4	4	1	≤0.5	83	F	4-Dec	A2	258
KPC-negative strains (5)
A	S. marcescens	2007	2	2	2	0.5	≤1	2	1	4	≤1	0.12	>8	0.5	>4	NA	NA	NA
B	E. aerogenes	2010	4	8	>64	>16	>32	>32	>16	2	>8	>4	>8	0.25	≤0.5	67	M	4-Dec
B	K. pneumoniae	2007	1	2	>64	>16	>32	>32	>16	32	>8	>4	≤1	0.12	≤0.5	57	F	5-Dec
B	K. pneumoniae	2008	1	2	>64	>16	>32	>32	>16	32	>8	>4	4	0.5	≤0.5	58	F	5-Dec
B	K. pneumoniae	2009	2	4	>64	>16	>32	>32	>16	1	>8	>4	>8	0.5	>4	53	F	15-Sep

IPM, imipenem; MER, meropenem; P/T, piperacillin/tazobactam; AZT, aztreonam; CAZ, ceftazidime; CRO, ceftriaxone; CPM, cefepime; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin; TET, tetracycline; TGC, tigecycline; COL, colistin; NA, not available; PFGE, pulsed-field gel electrophoresis.

Isolates were screened for carbapenemase-encoding genes (bla_IMP, bla_VIM, bla_SPM-1, bla_KPC, bla_SME, bla_IMI, bla_NMC-A, bla_GES, and bla_OXA-48) by standard PCR and amplicons were subjected to sequencing. Nucleotides and deduced amino acid sequences were analyzed using the Lasergene software package (DNASTAR, Madison, WI). Sequences were compared to others available in NCBI BLAST. bla_KPC-2 was detected in 18 (2.0% of total) isolates (17 K. pneumoniae and one C. freundii) and these strains were collected in 2008 (3/150 strains; 2.0%) and 2010 (15/289 strains; 5.1%; Table 1).

KPC-2-producing K. pneumoniae isolates were evaluated using pulsed-field gel electrophoresis (PFGE) and at least one representative strain of each PFGE type was selected for MLST according to the instructions on the website www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html. Isolates clustering within PFGE A were collected from hospital B in 2008 (two strains) and 2010 (six strains) and 5/6 isolates showing profile A1 were colistin resistant (MIC,>4 μg/ml, Table 1). Five KPC-2 producers grouped within PFGE type B and were collected during 2010 from both hospitals A and B. Two other genetically related strains (PFGE type E) were collected from hospital B (2010) and the two remaining strains each from hospital A and B had unique PFGE patterns (C and D, 2010). Strains displaying PFGE type A and E were associated to ST258, while those showing PFGE types B, C, and D were ST307, ST901, and ST494, respectively (Table 1).

The genetic location of bla_KPC-2 was evaluated using partial digestion with S1 nuclease and ICeuI digestion of total bacterial DNA resolved by electrophoresis followed by Southern blotting and hybridization with the bla_KPC-specific probe. All KPC-2 producers carried at least two plasmids and strains showing PFGE patterns A1-3, B1, C, and E possessed very similar bla_KPC-2-harboring plasmids in size (ca 125 kb). Similarly, strains displaying PFGE profiles B3 and B2 had a bla_KPC-2-carrying plasmid of ca 180 kb, while K. pneumoniae with PFGE patterns B4 and D showed smaller bla_KPC-2 plasmid bands (ca. 50-kb). The bla_KPC-2-carrying C. freundii appeared to possess three large plasmid bands of ca 200, 290, and 400 kb. Hybridization signals were observed in the 200- and 400-kb bands.

The bla_KPC-carrying element (Tn4401) was amplified with several primers and PCR products digested with Eag I. Those amplicons exhibiting an unusual digestion profile were sequenced. One and 14 strains carried bla_KPC-2 on copies of Tn4401b and Tn4401a, respectively. Three strains had differences in the genetic structure carrying the carbapenemase gene, two carried a Tn3 transposase upstream of bla_KPC-2, and the other had two adjacent copies of bla_KPC.

Among the clinical isolates from three medical institutions in Texas submitted as part of the SENTRY Program, KPC-producing strains were not observed before 2008, when three (2.0%) bla_KPC-2-carrying strains were detected, followed by an increase in the number of KPC-producing isolates in 2010 (15/289 [5.1%]; p=0.11; odds ratio [OR]=0.37 [0.08–1.40]). The increase in number of KPC-2 producers was due to the spread of two clonal clusters (PFGE type A and B) associated with different lineages of K. pneumoniae (ST258 and ST307). Plasmid dissemination also seemed to have played a role regarding the spread of bla_KPC-2, since similar bla_KPC-2-harboring plasmids were detected in strains from distinct PFGE and MLST types. The prevalence of KPC-2 producers in Texas remains low; however, this investigation shows a trend toward increased rates over the study interval (2007–2010), warranting continued surveillance and implementation of infection control measures to minimize the dissemination of carbapenem-resistant strains.

Disclosure Statement

JMI Laboratories, Inc. (MC, SEF, RNJ, and REM) has received research and educational grants in 2009–2012 from American Proficiency Institute (API), Anacor, Astellas, AstraZeneca, Bayer, Cempra, Cerexa, Contrafect, Cubist, Daiichi, Dipexium, Enanta, Furiex, GlaxoSmithKline, Johnson & Johnson (Ortho McNeil), LegoChem Biosciences, Inc., Meiji Seika Kaisha, Merck, Nabriva, Novartis, Pfizer (Wyeth), Rempex, Rib-X Pharmaceuticals, Seachaid, Shionogi, The Medicines Co., Theravance, ThermoFisher, and some other corporations. Some JMI employees are advisors/consultants for Astellas, Cubist, Pfizer, Cempra, Cerexa-Forest, J&J, and Theravance. In regard to speakers, bureaus, and stock options, none to declare. AW: none to declare. KVR: none to declare.

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