High Prevalence of CTX-M-15-Type Extended-Spectrum β-Lactamase Among AmpC β-Lactamase-Producing Klebsiella pneumoniae Isolates Causing Bacteremia in Korea

Abstract

We investigated the antimicrobial susceptibility, the genotypic distributions of extended-spectrum β-lactamase (ESBL) and AmpC genes, and the molecular epidemiology of AmpC-producing Klebsiella pneumoniae (AmpC-KP) isolates causing bacteremia. Among 260 K. pneumoniae clinical isolates included in this study, plasmid-mediated AmpC β-lactamases were found in 20.7% (54/260), which included DHA-1 (96.3%, 52/54), CMY-2 (3.7%, 2/54), and CMY-10 (1.9%, 1/54). One isolate also produced DHA-1 along with CMY-2. Of the 54 AmpC-KP isolates, 31 isolates (57.4%) showed ESBL positivity. Of these 31 isolates with coproduction of ESBL and AmpC β-lactamases, 25 isolates (80.6%) produced CTX-M-15 in addition to DHA-1. Nine isolates (29.0%) were nonsusceptible to imipenem. The most prevalent sequence type (ST) was ST11 (n = 31, 57.4%), followed by ST2361 (n = 5, 9.3%), which was newly identified in this study, and ST48 (n = 4, 7.4%). K. pneumoniae isolates coproducing DHA-1 and CTX-M-15 have emerged and disseminated in Korean hospitals, even in blood isolates causing bacteremia. Such infections may become a challenge for clinicians because there is a severely restricted range of available therapeutic options for these pathogens.

Introduction

Enterobacteriaceae expressing the plasmid AmpC β-lactamases have become a major clinical concern, as these enzymes confer resistance to β-lactam antibiotics, particularly cephalosporins, and may be accompanied by coresistance to drugs of other classes.¹ Plasmid-mediated AmpC (pAmpC) β-lactamases have arisen through the transfer of chromosomal genes encoding inducible AmpC β-lactamase onto plasmids. This transfer has resulted in pAmpC strains, especially in Klebsiella pneumoniae, which naturally lack chromosomal-mediated AmpC β-lactamases. Similar to extended-spectrum β-lactamases (ESBLs), pAmpC β-lactamases have broad substrate profiles that include penicillin, cephalosporins, and monobactams.² Consequently, both ESBL and pAmpC β-lactamase are typically associated with broad multidrug resistance (MDR) because multiple antibiotic resistance genes exist on the same plasmid.³ More recently, gram-negative organisms that produce both ESBL and pAmpC β-lactamase have increasingly been described worldwide. Several studies have focused on the clinical features of ESBL- or AmpC-producing Enterobacteriaceae, but molecular characterization data are still lacking. In the present study, we report the high prevalence of CTX-M-15 among AmpC β-lactamase-producing K. pneumoniae isolates causing bacteremia. We assessed the in vitro antimicrobial susceptibility and genotypic distribution of AmpC and ESBL coproducing K. pneumoniae blood isolates in various regions of Korea.

Materials and Methods

Screening of AmpC β-lactamases and ESBL-producing isolates

A total of 200 K. pneumoniae clinical isolates causing bacteremia were collected from January 2010 to December 2014 at Samsung Medical Center (Seoul, Korea). In addition, 60 isolates were included from isolates in the Asian Bacterial Bank (Asia Pacific Foundation for Infectious Diseases, Seoul, Korea), which had been collected during bacteremia studies in Korea between 2012 and 2013. These clinical isolates were conventionally identified using VITEK2 systems (BioMeriéux, France) in the clinical microbiology laboratories in each hospital. Only the first isolate per patient was included in the study.

Cefoxitin nonsusceptible isolates were detected by the VITEK system. Isolates showing cefoxitin nonsusceptibility (≥16 mg/L), which were detected by the VITEK system, were considered presumptive AmpC producers. ESBL activity was confirmed via a double-disk synergy test (ceftazidime, cefotaxime, and aztreonam minimum inhibitory concentration [MIC] of ≥2 mg/L) using BD BBL Sensi-Disks (BD Diagnostics, Sparks, MD).

Antimicrobial susceptibility testing

For all the AmpC-PCR positive isolates, susceptibility testing was conducted, using the broth microdilution method and the Etest.⁴ Eight antimicrobial agents were tested: ceftazidime, cefotaxime, cefepime, piperacillin–tazobactam, ciprofloxacin, imipenem, meropenem, and ertapenem. MICs were interpreted with category designations according to the criteria of the CLSI guidelines.⁴ Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as control strains.

Detection of β-lactamase genes and multilocus sequence typing

Cefoxitin nonsusceptible isolates were tested by PCR amplification and DNA sequencing for various pAmpC β-lactamase genes.^5,6 For the detection of ESBL, if the screening test result was positive, the type of the β-lactamase was investigated as described.⁵ The genetic relationship of AmpC and ESBL coproducing isolates was assessed using multilocus sequence typing (MLST) as described previously.⁷ New STs were subsequently submitted to the MLST website and approved.

Determination of plasmid replicon type

AmpC-producing K. pneumoniae (AmpC-KP) isolate plasmid replicon types were identified using PCR-based replicon typing methods, which include five different multiplex PCRs and three simplex PCRs.⁸ Replicon types were identified by sequencing PCR products.

Results and Discussion

pAmpC β-lactamases were found in 54 (20.7%) of 260 K. pneumoniae isolates causing bacteremia. Of these AmpC-KP isolates, more than 60% of the isolates were nonsusceptible to ceftazidime, cefotaxime, cefepime, piperacillin–tazobactam, and ciprofloxacin (Table 1). In addition, AmpC and ESBL coproducing isolates showed a high frequency of coresistance to ceftazidime, cefotaxime, cefepime, piperacillin–tazobactam, and ciprofloxacin. Among the carbapenems, the antimicrobial nonsusceptible rate was the highest for ertapenem in both AmpC-producing and AmpC and ESBL coproducing isolates (35.2% and 29.0%, respectively). Nonsusceptibility to carbapenems in the AmpC and ESBL coproducing K. pneumoniae isolates was rather low compared with AmpC-KP. The most prevalent AmpC β-lactamase type was DHA-1 (n = 52, 96.3%). CMY group genes were also detected in three isolates, which included CMY-2 (n = 2, 3.7%) and CMY-10 (n = 1, 1.9%), respectively. One isolate also produced DHA-1 along with CMY-2.

Table 1.

Results of Antimicrobial Susceptibility Testing of AmpC-Producing and AmpC and Extended-Spectrum β-Lactamases Coproducing Klebsiella pneumoniae Isolates Causing Bacteremia

	AmpC-producing K. pneumoniae (n = 54)			AmpC and ESBL coproducing K. pneumoniae (n = 31)
	MIC (mg/L) ^a			MIC (mg/L)
Antimicrobial agents	50%	90%	No. (%) of nonsusceptible isolates	50%	90%	No. (%) of non-susceptible isolates
Ceftazidime	128	>128	53 (98.1)	128	>128	31 (100)
Cefotaxime	>128	>128	52 (96.3)	>128	>128	31 (100)
Cefepime	32	>128	34 (63.0)	32	>128	23 (74.2)
Piperacillin/tazobactam	>256/4	>256/4	41 (75.9)	>256/4	>256/4	24 (77.4)
Ciprofloxacin	>64	>64	51 (94.4)	>64	>64	29 (93.5)
Imipenem	1	8	13 (24.1)	1	8	6 (19.4)
Meropenem	0.06	4	7 (13.0)	0.06	4	3 (9.7)
Ertapenem	0.5	32	19 (35.2)	0.5	32	9 (29.0)

50% and 90%, MICs at which 50% and 90% of the isolates, respectively, were inhibited.

ESBL, extended-spectrum β-lactamase; MIC, minimum inhibitory concentration.

Among the 54 AmpC-producing isolates, 47 (87.0%) possessed SHV-type genes. As expected, non-ESBL SHV-11 (n = 26, 48.1%) was the most common type, followed by non-ESBL SHV-1 (n = 15, 27.8%), SHV-187 (n = 2, 3.7%), and SHV-2A (n = 1, 1.9%). The most prevalent ESBL gene was CTX-M-15 (n = 26, 48.1%), followed by CTX-M-14 (n = 6, 11.1%), and CTX-M-3 (n = 2, 3.7%). Four isolates also produced CTX-M-14 along with CTX-M-15. Taken together, of the AmpC-KP, 31 isolates (57.4%) also produced ESBL, including those that were CTX-M-type (n = 30, 55.6%) and SHV-12 (n = 1, 1.8%). Of these, 25 isolates (80.6%) produced CTX-M-15 in addition to DHA-1.

ST11 (n = 31, 57.4%) was the most prevalent clone in AmpC-KP isolates (Table 2). The second most prevalent clone was ST2361 (n = 5, 9.3%) and all the ST2361 isolates also produced DHA-1 along with CTX-M-15 and SHV-1. Except for ST11, ST2361, and ST48, most STs were represented by one to three K. pneumoniae isolates. IncFIIA (n = 19, 35.2%) was the most frequently detected plasmid replicon type, followed by IncF (n = 11, 20.4%). Four isolates (7.4%) had IncF together with IncFIIA and one isolate (1.9%) had IncF together with IncA/C. In ST11 strains, 12 of the 31 isolates (38.7%) harbored plasmid replicon type IncFIIA.

Table 2.

Multilocus Sequence Typing Analysis of AmpC-Producing Klebsiella pneumoniae Isolates

		β-lactamase type
Sequence type (No. of isolates)	Total (n = 54)	AmpC (n = 54)	CTX-M (n = 30)	Others ^b (No. of isolates)
ST11 (31)	15	DHA-1		SHV-1 (1), SHV-11 (9)
	11	DHA-1	CTX-M-15	TEM-1+SHV-11 (7)
	11	DHA-1	CTX-M-15	SHV-12 (1)
	2	DHA-1	CTX-M-14	SHV-2a (1), SHV-12 (1)
	1	DHA-1	CTX-M-15 + 14	SHV-11 (1)
	1	DHA-1	CTX-M-3	SHV-11 (1)
	1	CMY-10	CTX-M-3	TEM-1+SHV-11 (1)
ST2361 (5)^a	5	DHA-1	CTX-M-15	SHV-1 (5)
ST48 (4)	2	DHA-1	CTX-M-15	SHV-1 (2)
	1	DHA-1	CTX-M-15 + 14	SHV-1 (1)
	1	DHA-1		SHV-1 (1)
ST307 (2)	1	DHA-1	CTX-M-15	SHV-1 (1)
	1	DHA-1		SHV-1 (1)
ST711 (2)	2	DHA-1	CTX-M-15	TEM-1+SHV-187 (2)
Others (10)	10

New sequence type identified in this study.

bla_TEM-1, bla_SHV-1, and bla_SHV-11 are non-ESBL genes as reference.

ST, sequence type.

The therapeutic options for ESBL-producing organisms are very limited. Moreover, the coexistence of different classes of β-lactamases in a single bacterial isolate can cause treatment failure because these combinations severely restrict the range of available therapeutic options.⁹

Our findings showed that 57.4% of the pAmpC-producing K. pneumoniae blood isolates also produced ESBL. DHA-1 was the most prevalent AmpC gene and predominantly presented in CTX-M-15-producing K. pneumoniae isolates. CTX-M-15 was the most prevalent ESBL detected, whereas the previous study reported that SHV-12 was the most common gene cluster of ESBL in our region.¹⁰ As expected, ESBL and AmpC-β-lactamase-producing isolates were highly resistant to all cephalosporins, piperacillin–tazobactam, and ciprofloxacin. The carbapenems were generally the most effective agents against both ESBL and AmpC β-lactamase producers.

ST11, which is the major clone of KPC-producing K. pneumoniae in Asia,¹¹ was the most prevalent clone in our study. Among 25 DHA-1 and CTX-M-15 producers, 12 isolates (48%) belonged to ST11. The propensity of ST11 to acquire multiple resistance mechanisms greatly enhances its epidemic potential.¹² Another prevalent ST, ST2361, was first identified in this study. ST2361 is a single locus variant of ST48 differing at the tonB locus. PCR-based replicon typing showed that IncFIIA replicons were prevalent in DHA-1 and CTX-M-15 coproducing K. pneumoniae isolates (n = 19, 35.2%). It has been proposed that the horizontal transfer of bla_CTX-M-15 from E. coli to K. pneumoniae resulted from conjugation of IncFII plasmids.¹³ Although relatively small sample size could be included and the distribution of isolates was not balanced among the different hospitals, our results showed that most bla_CTX-M-15 genes are mainly associated with the IncFIIA plasmid, consistent with previous studies.^14,15

Carbapenems are first-choice drugs for the treatment of ESBL or AmpC-β-lactamase-producing pathogens, especially with increasing reports of ESBL-producing clinical isolates expressing MDR.^16,17 A worrisome aspect of the rise of carbapenem-resistant pathogens is their ability to exchange genes with coresiding bacteria through horizontal gene transfer via plasmids.¹⁸ Incorporation of emerging resistance determinants within CTX-M-producing bacteria, such as those encoding carbapenemases, could be a consequence of the failure to control the spread of these strains.¹⁹

Given the relatively small number of cases, there is the possibility of false-negative results because the identification of pAmpC producers is challenging and missed isolates cannot be excluded.²⁰ There is a constant risk that further increases in prevalence might go undetected. A large epidemiological study is needed to more fully understand the prevalence of ESBL and pAmpC-producing strains in a hospital setting.

K. pneumoniae isolates coproducing DHA-1 and CTX-M-15 have emerged and disseminated throughout Korean hospitals. The high prevalence of DHA-1 and CTX-M-15-producing K. pneumoniae isolates may be due to a combination of the frequent incorporation of many IncFIIA-type plasmids and the clonal spread of certain clones such as ST11. Such infections may become a challenge for clinicians because there is a severely restricted range of available therapeutic options for these pathogens. Enhanced infection control measures should be implemented to prevent their further dissemination.

Footnotes

Acknowledgments

The authors thank all participating investigators in the Korean Network for Study on Infectious Diseases (KONSID). The members who participated in this study in the KONSID are as follows: Yu Mi Wi (Samsung Changwon Hospital, Changwon, Korea); Jun Seong Son and Soo-Youn Moon (Kyung Hee University Hospital at Gangdong, Seoul, Korea); Seong Yeol Ryu and Hyun Ah Kim (Keimyung University Dongsan Medical Center, Daegu, Korea); Ki Tae Kwon (Daegu Fatima Hospital, Daegu, Korea); Min Hee Lim (Changwon Fatima Hospital, Changwon, Korea); and Yeon-Sook Kim and Kyung Mok Sohn (Chungnam National University Hospital, Daejeon, Korea).

Bacterial isolates were obtained from the Asian Bacterial Bank (ABB) of the Asia Pacific Foundation for Infectious Diseases (APFID).

Disclosure Statement

No competing financial interests exist.

References

Jacoby

G.A.

2009. AmpC beta-lactamases. Clin. Microbiol. Rev., 22:161–182.

Black

J.A.

, Moland

E.S.

, and Thomson

K.S.

. 2005. AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J. Clin. Microbiol., 43:3110–3113.

Yoon

Y.K.

, Cheong

H.W.

, Pai

, Roh

K.H.

, Kim

J.Y.

, Park

D.W.

, Sohn

J.W.

, Lee

S.E.

, Chun

B.C.

, Sim

H.S.

, and Kim

M.J.

. 2011. Molecular analysis of a prolonged spread of Klebsiella pneumoniae co-producing DHA-1 and SHV-12 beta-lactamases. J. Microbiol., 49:363–368.

CLSI. 2015. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fifth Informational Supplement. M100-S25. Clinical and Laboratory Standards Institute, Wayne, PA.

Cohen Stuart

, Dierikx

, Al Naiemi

, Karczmarek

, Van Hoek

A.H.

, Vos

, Fluit

A.C.

, Scharringa

, Duim

, Mevius

, and Leverstein-Van Hall

M.A.

. 2010. Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. J. Antimicrob. Chemother., 65:1377–1381.

Dallenne

, Da Costa

, Decre

, Favier

, and Arlet

. 2010. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother., 65:490–495.

Diancourt

, Passet

, Verhoef

, Grimont

P.A.

, and Brisse

. 2005. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J. Clin. Microbiol., 43:4178–4182.

Carattoli

, Bertini

, Villa

, Falbo

, Hopkins

K.L.

, and Threlfall

E.J.

. 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods, 63:219–228.

Song

, Kim

J.S.

, Kim

H.S.

, Yong

, Jeong

S.H.

, Park

M.J.

, and Lee

K.M.

. 2006. Increasing trend in the prevalence of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal ampC gene at a Korean university hospital from 2002 to 2004. Diagn. Microbiol. Infect. Dis., 55:219–224.

10.

K.S.

, Lee

J.Y.

, Baek

J.Y.

, Suh

J.Y.

, Lee

M.Y.

, Choi

J.Y.

, Yeom

J.S.

, Kim

Y.S.

, Jung

S.I.

, Shin

S.Y.

, Heo

S.T.

, Kwon

K.T.

, Son

J.S.

, Kim

S.W.

, Chang

H.H.

, Ki

H.K.

, Chung

D.R.

, Peck

K.R.

, and Song

J.H.

. 2010. Predominance of an ST11 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae clone causing bacteraemia and urinary tract infections in Korea. J. Med. Microbiol., 59:822–828.

11.

Andrade

L.N.

, Curiao

, Ferreira

J.C.

, Longo

J.M.

, Climaco

E.C.

, Martinez

, Bellissimo-Rodrigues

, Basile-Filho

, Evaristo

M.A.

, Del Peloso

P.F.

, Ribeiro

V.B.

, Barth

A.L.

, Paula

M.C.

, Baquero

, Canton

, Darini

A.L.

, and Coque

T.M.

. 2011. Dissemination of blaKPC-2 by the spread of Klebsiella pneumoniae clonal complex 258 clones (ST258, ST11, ST437) and plasmids (IncFII, IncN, IncL/M) among Enterobacteriaceae species in Brazil. Antimicrob. Agents Chemother., 55:3579–3583.

12.

Luk

, Wong

W.K.

, Ho

A.Y.

, Yu

K.C.

, To

W.K.

, and Ng

T.K.

. 2016. Clinical features and molecular epidemiology of plasmid-mediated DHA-type AmpC beta-lactamase-producing Klebsiella pneumoniae blood culture isolates, Hong Kong. J. Glob. Antimicrob. Resist., 7:37–42.

13.

Diestra

, Juan

, Curiao

, Moya

, Miro

, Oteo

, Coque

T.M.

, Perez-Vazquez

, Campos

, Canton

, Oliver

, and Navarro

. 2009. Characterization of plasmids encoding blaESBL and surrounding genes in Spanish clinical isolates of Escherichia coli and Klebsiella pneumoniae. J. Antimicrob. Chemother., 63:60–66.

14.

Gonullu

, Aktas

, Kayacan

C.B.

, Salcioglu

, Carattoli

, Yong

D.E.

, and Walsh

T.R.

. 2008. Dissemination of CTX-M-15 beta-lactamase genes carried on Inc FI and FII plasmids among clinical isolates of Escherichia coli in a university hospital in Istanbul, Turkey. J. Clin. Microbiol., 46:1110–1112.

15.

Coelho

, Gonzalez-Lopez

J.J.

, Miro

, Alonso-Tarres

, Mirelis

, Larrosa

M.N.

, Bartolome

R.M.

, Andreu

, Navarro

, Johnson

J.R.

, and Prats

. 2010. Characterisation of the CTX-M-15-encoding gene in Klebsiella pneumoniae strains from the Barcelona metropolitan area: plasmid diversity and chromosomal integration. Int. J. Antimicrob. Agents, 36:73–78.

16.

Morosini

M.I.

, Garcia-Castillo

, Coque

T.M.

, Valverde

, Novais

, Loza

, Baquero

, and Canton

. 2006. Antibiotic coresistance in extended-spectrum-beta-lactamase-producing Enterobacteriaceae and in vitro activity of tigecycline. Antimicrob. Agents Chemother., 50:2695–2699.

17.

Kang

C.I.

, and Song

J.H.

. 2013. Antimicrobial resistance in Asia: current epidemiology and clinical implications. Infect. Chemother., 45:22–31.

18.

Magiorakos

A.P.

, Srinivasan

, Carey

R.B.

, Carmeli

, Falagas

M.E.

, Giske

C.G.

, Harbarth

, Hindler

J.F.

, Kahlmeter

, Olsson-Liljequist

, Paterson

D.L.

, Rice

L.B.

, Stelling

, Struelens

M.J.

, Vatopoulos

, Weber

J.T.

, and Monnet

D.L.

. 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect., 18:268–281.

19.

Canton

, Gonzalez-Alba

J.M.

, and Galan

J.C.

. 2012. CTX-M Enzymes: origin and Diffusion. Front. Microbiol., 3:110.

20.

Conen

, Frei

, Adler

, Dangel

, Fux

C.A.

, and Widmer

A.F.

. 2015. Microbiological screening is necessary to distinguish carriers of plasmid-mediated AmpC beta-lactamase-producing enterobacteriaceae and extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae because of clinical similarity. PLoS One, 10:e0120688.