Emergence of serotype K1 Klebsiella pneumoniae ST23 strains co-producing the plasmid-mediated AmpC beta-lactamase DHA-1 and an extended-spectrum beta-lactamase in Korea

Serotype K1 Klebsiella pneumoniae has emerged as an important community pathogen causing various infections, including liver abscesses. Although serotype K1 K. pneumoniae community isolates have been reported as susceptible to most classes of antimicrobial agents, a few cases of infection caused by extended-spectrum beta-lactamase (ESBL)-producing serotype K1 K. pneumoniae have recently been reported in Asian countries. We identified three ESBL-producing strains of serotype K1 K. pneumoniae and conducted a molecular characterization of their drug resistance. Three ESBL-producing serotype K1 K. pneumoniae ST23 strains were identified from strains in the Asian Bacterial Bank. Antimicrobial susceptibility testing was performed using the broth microdilution method, and ESBL production was tested by the double-disk synergy test and a confirmatory test. PCR was performed to detect the genes for plasmid-mediated ESBL and AmpC beta-lactamases. All three strains were resistant to cefotaxime, ceftazidime, and piperacillin/tazobactam, and all were determined to be ESBL-producers. No known ESBL genes, including blaSHV, blaTEM, blaCTX-M, blaGES, blaPER, and blaVEB, were detected among the three strains. Of all plasmid-mediated AmpC beta-lactamase (PAB) genes, including blaDHA-1, blaCMY, blaFOX, and blaMOX, the blaDHA-1 gene was detected in two of the strains. The PFGE patterns revealed that the two isolates carrying blaDHA-1 were closely related (84% similarity). No ESBL genes were detected among three ESBL-producing serotype K1 K. pneumoniae ST23 strains. Two strains contained the PAB gene blaDHA-1. The emergence of resistant strains of community-origin serotype K1 K. pneumoniae has important implications for effective treatment and infection control practices.


Background
The rates of extended-spectrum beta-lactamase (ESBL)production in Klebsiella pneumoniae are very high in hospitals worldwide, and highly resistant strains such as K. pneumoniae carbapenemase (KPC) or New Delhi metallo-beta-lactamase (NDM)-producing K. pneumoniae have been rapidly spreading between countries. Treatment of infections caused by multidrug-resistant K. pneumoniae has been a challenge, and there is increasing concern about the economic impact of these bacteria.
Serotype K1 K. pneumoniae has been reported as the predominant serotype among those isolates causing liver abscesses [1,2]. Most K1 strains isolated from liver abscess cases belong to sequence type (ST) 23 in Asian countries [3,4]. Although ESBL K. pneumoniae and KPC-producing K. pneumoniae have become globally widespread, a distinctive characteristic of K1 K. pneumoniae ST23 has been good susceptibility to most antibiotic classes (except ampicillin and piperacillin) [5], however, ESBL-producing K1 K. pneumoniae strains have begun to be reported recently [6,7]. In this study, we report serotype K1 K. pneumoniae strains co-producing the plasmid-mediated AmpC beta-lactamase (PAB) DHA-1 and ESBL.

Materials and methods
Three ESBL-producing serotype K1 K. pneumoniae ST23 strains were identified from strains in the Asian Bacterial Bank (Asia Pacific Foundation for Infectious Diseases, Seoul, Korea), which had been collected during bacteremia studies in Korea between 2006 and 2008.
Double-disk synergy test-positive isolates were further tested by polymerase chain reaction (PCR) and sequence analyses to determine the gene responsible for the ESBL phenotype in the ESBL producers. In the ESBL-producing K. pneumoniae isolates, bla TEM , bla SHV , bla CTX-M , bla GES , bla PER , and bla VEB genes were tested by PCR. In addition, bla DHA-1 , bla CMY , bla FOX , bla MOX, bla IMP , bla VIM , bla OXA , and bla NDM genes were tested. The sequences were compared with those in the GenBank nucleotide database for subtyping.
To determine serotype K1, PCR was conducted using a primer pair specific for magA, which is a gene specific for the K1 antigen. The primers were chosen as previously described: forward, 5′GGTGCTCTTTACATCATTGC-3′, and reverse, 5′-GCAATGGCCATTTGCGT TAG-3′ [9]. Multilocus sequence typing (MLST) was conducted using the nucleotide sequences of seven housekeeping genes (gagA, infB, mdh, pgi, phoE, rpoB, and tonB) as previously described [10]. For pulsed field gel electrophoresis (PFGE), agarose-embedded bacterial genomic DNA was digested with 20 U of XbaI. The restriction fragments were separated by gel electrophoresis in 0.5 × Tris-borate-EDTA buffer. Electrophoresis was performed using a CHEF Mapper XA (Bio-Rad Laboratories, Hercules, CA, USA). The PFGE patterns were analyzed using GelCompar II version 6.1 (Applied Maths, Belgium).

Results
Of the 120 K. pneumoniae isolates, 20 (16.7%) were ESBLproducers. Among the ESBL-producers, only three were determined to be serotype K1 (KPN1, KPN2, and KPN3). Antimicrobial susceptibilities of the three isolates are shown in Table 1. All three strains were resistant to ampicillin, piperacillin-tazobactam, cefotaxime, ceftazidime, and rifampin. Two strains (KPN2 and KPN3) were also resistant to amikacin and ciprofloxacin. One strain (KPN2) was resistant to imipenem. In all three strains, no ESBL genes, including bla TEM , bla SHV , bla CTX-M , bla GES , bla PER , and bla VEB , were detected. Among the PAB genes (bla DHA-1 , bla CMY , bla FOX , and bla MOX ), only bla DHA-1 was detected in two strains (KPN2 and KPN3). Carbapenemase genes were also not detected in all three isolates ( Table 2). Clonal relatedness was investigated by PFGE for the two strains. Analysis of the PFGE patterns showed that the two isolates carrying bla DHA-1 were closely related (84% similarity).

Discussion
Serotype K1 K. pneumoniae ST23 is, a highly virulent pathogenic strain causing invasive community-acquired infections, that is widespread in its geographical distribution in Asia [5]. Fortunately, ST23 strains have shown good susceptibility to most antibiotics. However, the advent of multidrug resistance with high transmission potential in K. pneumoniae serotype K1 causes serious concerns. A few strains of ESBL-producing serotype K1 K. pneumoniae have already been reported in Asian countries [6,7,11]. In the present study, we report serotype K1 K. pneumoniae strains co-producing the PAB DHA-1 and ESBL. We found three isolates that were phenotypically ESBL producers, but we could not detect any bla genes responsible for the ESBL phenotype. Therefore, other ESBL genotypes may be involved. In addition, two of the three isolates were identified as PAB. Recently, gram-negative organisms that produce both ESBLs and PAB enzymes have increasingly been described worldwide [12][13][14][15]. To our knowledge, this is the first report of serotype K1 K. pneumoniae producing both ESBL and PAB. Both ESBLs and PAB enzymes are associated with broad, multidrug resistance because multiple antibiotic-resistance genes exist on the same plasmid [16]. One isolate (KPN3) in our study showed carbapenem resistance in the broth microdilution test. Subsequently, we used PCR analysis to determine the presence of carbapenemase genes, such as bla IMP , bla VIM , bla OXA , and bla NDM ; however, none of these genes were detected. The frequency of PAB transmission may be higher than initially thought, especially if the spread of resistance mimics the trend that we have seen occurring over the past few years for ESBLs. Moreover, porin alteration, combined with the production of ESBL or PAB, has been demonstrated to confer carbapenem resistance [17,18]. We also characterized antimicrobial susceptibility between non-serotype K1 and serotype K1 ESBL-producing K. pneumoniae. Although the number of total isolates was too small to draw definite conclusions, the resistant rates of piperacillin-tazobactam and imipenem in K1 isolates tended to be high. It is uncertain whether the increase of antimicrobial resistance in serotype K1 K. pneumoniae is connected with any one particular resistance gene.
The emergence of multidrug-resistant strains with high transmission potential in serotype K1 K. pneumoniae is of great concern due to limited alternative treatment options and the possibility of global dissemination. Careful surveillance of resistant strains and adequate infection prevention and control measures are necessary.   TEM SHV CTX-M PER KPC PER VEB GES/IBC SME IMI/NMC CMY ACT FOX DHA  ACC MOX IMP VIM OXA NDM   KPN1 --b  ----------------- TEM-1 (not an ESBL gene), b SHV-11 (not an ESBL gene)