Transfer of CMY-2 Cephalosporinase from Escherichia coli to Virulent Klebsiella pneumoniae Causing a Recurrent Liver Abscess

Abstract

A CMY-2-producing capsular type K2 Klebsiella pneumoniae strain (TVGHKP93) with multidrug resistance was isolated from a recurrent liver abscess in a patient who also carried a CMY-2-producing Escherichia coli strain (TVGHEC01) in the stool. TVGHKP93 retained its high virulence compared with that of the isogenic strain (TVGHKP60) with wild-type resistance from the first liver abscess. Our conjugation experiment showed the successful transfer of the bla_CMY-2-carrying plasmid from TVGHEC01 into TVGHKP60. The transconjugant showed both high virulence and the multidrug-resistant phenotype, as did TVGHKP93.

TEXT

Klebsiella pneumoniae liver abscess (KPLA) has been increasingly reported in Asia and is considered to be endemic in Taiwan (1, 2). The capsular type of K. pneumoniae appears to be the major virulence factor (3, 4), and the K1 and K2 types were the most prevalent in KPLA (1). In KPLA, K. pneumoniae isolates demonstrate an almost unique antibiogram indicative of resistance to ampicillin only, and multidrug-resistant isolates have rarely been reported (5, 6). Currently, several investigations have provided evidence that KPLA is preceded by gastrointestinal colonization (7–10).

We identified an 84-year-old patient with diabetes who had recurrent KPLA. The strain (TVGHKP60) isolated from the first abscess in November 2012 was susceptible to all antibiotics tested, except for ampicillin, consistent with the natural resistance of K. pneumoniae. The patient received intravenous ceftriaxone for 3 weeks and recovered well. After discharge, he received oral cefuroxime for another 2 weeks. However, the patient had a recurrent liver abscess in January 2013, and a second K. pneumoniae strain with multidrug resistance (TVGHKP93) was isolated. The patient received intravenous ciprofloxacin for 3 weeks and was discharged uneventfully. Interestingly, a multidrug-resistant Escherichia coli strain (TVGHEC01) was isolated from the patient's stool during the recurrence episode. We further investigated the two K. pneumoniae strains and one E. coli strain from this case. The protocol was approved by the institutional review board of Taipei Veterans General Hospital.

Bacterial identification and antimicrobial susceptibility were determined using a Vitek2 system (bioMérieux, Marcy l'Etoile, France). The antimicrobial susceptibility was interpreted according to the guidelines of the CLSI (11) and is shown in Table 1. Pulsed-field gel electrophoresis DNA fingerprinting (12, 13) showed that the wild-type TVGHKP60 strain was nearly identical (only one band difference) to the multidrug-resistant TVGHKP93 strain. Capsular genotyping, detection of rmpA/rmpA2, molecular characterization of β-lactamases, and colony mucoviscosity were performed as previously described (14–16). Multilocus sequence typing (MLST) was performed on the TVGHKP60 and TVGHKP93 strains, and the results were analyzed as previously described (17). The TVGHKP60 and TVGHKP93 strains both belonged to capsular type K2 and sequence type (ST) 86. They showed hypermucoviscosity phenotypes and carried rmpA and rmpA2 genes. Regarding the detection of β-lactamases, bla_SHV-1 was detected in the TVGHKP60 strain, and bla_SHV-1 and bla_CMY-2 were detected in the TVGHKP93 strain. Interestingly, bla_CMY-2 was also detected in the TVGHEC01 strain.

TABLE 1.

Antimicrobial susceptibility test data for the strains in this study

Antibiotic(s)	MIC (mg/liter)a
	K. pneumoniae TVGHKP60	K. pneumoniae TVGHKP93	E. coli TVGHEC01	K. pneumoniae transconjugant: TVGHKP60::blaCMY-2
Ampicillin	≥32	≥32	≥32	≥32
Cefazolin	≤4	≥64	≥64	≥64
Cefuroxime	2	16	16	16
Cefoxitin	≤4	32	32	32
Ceftriaxone	≤1	8	8	8
Ceftazidime	≤1	16	4	16
Cefepime	≤1	≤1	≤1	≤1
Piperacillin-tazobactam	≤4	8	≤4	8
Gentamicin	≤1	≤1	≤1	≤1
Amikacin	≤2	≤2	≤2	≤2
Ciprofloxacin	≤0.25	≤0.25	≤0.25	≤0.25
Levofloxacin	≤0.12	≤0.12	≤0.12	≤0.12
Ertapenem	≤0.5	≤0.5	≤0.5	≤0.5
Imipenem	≤0.25	≤0.25	≤0.25	≤0.25
Trimethoprim-sulfamethoxazole	0.19	0.38	0.094	0.38

^aThe values are MIC correlates determined by the Vitek2 system, except for trimethoprim-sulfamethoxazole, which was determined by an Etest.

The K. pneumoniae strains were cultured in LB broth at 37°C for 16 h to obtain bacterial growth curves as described previously (18). The isogenic bla_CMY-2-producing strain (TVGHKP93) and the original strain (TVGHKP60) showed no significant differences in their growth rates. The in vivo virulence of the TVGHKP60 and TVGHKP93 strains was compared using a murine model of septicemia generated by intraperitoneal injection. Male 6- to 8-week-old C57/B6 mice were observed for 1 week after intraperitoneal inoculation of 100 CFU of K. pneumoniae. All animal care procedures and protocols were approved by the Institutional Animal Care and Use Committee of National Yang-Ming University. Upon intraperitoneal infection of mice, both strains showed hypervirulence with 50% lethal dose(LD₅₀) values of <100 CFU (Fig. 1). The clinical K. pneumoniae strain (capsular type K64) isolated from blood was used as the control and its LD₅₀ was 10⁷ CFU.

FIG 1.

In vivo virulence study. Mouse lethality data following challenges with the TVGHKP60 and TVGHKP93 strains are presented. Male C57BL/6 mice (n = 6 from two independent experiments) were inoculated by intraperitoneal injection with 100 CFU of the TVGHKP60 and TVGHKP93 strains. Survival was assessed for 7 days following infection. The Kaplan-Meier method was used to evaluate the survival rate. Upon intraperitoneal infection, all mice from each group (TVGHKP60 versus TVGHKP93) were dead within 5 days (log rank test; P = 0.6285).

Plasmids were extracted from TVGHEC01 for high-throughput sequencing using the Illumina/Solexa GAII sequencing platform. Coding sequences were predicted and annotated with NCBI protein BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). A circular map of a bla_CMY-2-carrying plasmid, designated pCMY2, with 102,199 bp was obtained (GenBank accession no. LC019731). Nucleotide blast results revealed that pCMY2 exhibited a high level of similarity with a 108,660-bp E. coli plasmid, pC49-108 (GenBank accession no. KJ484638), which showed 94% coverage and shared 99% DNA sequence identity. Conjugation-related genes (associated with tra and trb) and type IV pili-associated genes were found in both plasmids. However, pC49-108 harbored the drug resistance genes addA5, dhfrA17, and bla_CTX-M-1, while pCMY2 carried an AmpC-type β-lactamase, bla_CMY-2, instead. The PCR results using four primer pairs located within the plasmid revealed that pCMY2 was present in both TVGHEC01 and TVGHKP93 but absent from the TVGHKP60. It is reasonable to consider that horizontal transfer of a bla_CMY-2-carrying resistance determinant from E. coli to K. pneumoniae developed in the patient's bowel.

Our attempts to mimic the natural transfer of the pCMY2 plasmid from TVGHEC01 into TVGHKP60 using conjugation experiments described previously (19) were successful. The conjugation frequency was determined to be 5.2 × 10⁻⁶. The MICs for cefazolin, cefuroxime, cefoxitin, ceftriaxone, ceftazidime, and piperacillin-tazobactam increased in the transconjugant (TVGHKP60::bla_CMY-2) and were the same as those for TVGHKP93 (Table 1). The in vivo virulence assessment also confirmed that K. pneumoniae TVGHKP60::bla_CMY-2 retained the hypervirulence characteristics (data not shown). These results implied that TVGHKP60 had acquired pCMY2 from TVGHEC01 in the patient's gut, leading to the formation of the hypervirulent and multidrug-resistant TVGHKP93 in the recurrent liver abscess.

Capsular type K2 and ST86 are considered to be hypervirulent clones that can cause invasive diseases (20, 21). Our study first demonstrated that this virulent strain acquired the pCMY2 plasmid but still retained its virulence. The selective pressure by cephalosporins may predispose to the possible plasmid transfer in this case. Although increased antimicrobial resistance is generally associated with decreased virulence and fitness, evidence has also shown the opposite, and it is increasingly evident that the relationship is often of greater benefit to the pathogen, resulting in a growing public health problem (22).

A recent study showed that multidrug-resistant and hypervirulent populations of K. pneumoniae were mostly nonoverlapping, although two isolates with combined virulence and resistance features were detected (23). These results show that the threat of dual-risk K. pneumoniae strains, combining virulence and multidrug-resistance features, is becoming a reality. The multidrug-resistant and highly virulent TVGHKP93 strain derived from the wild-type TVGHKP60 strain serves as a good example to verify the relationship between virulence and resistance in K. pneumoniae.

With the increasing rate of drug-resistant Enterobacteriaceae colonizing the intestine, the possibility of interspecies transfer of drug resistance determinants into highly virulent K. pneumoniae increases. The acquisition of an important mechanism of antibiotic resistance, such as CMY-2, might suggest that virulent strains may be a potential cause of nosocomial infections in the future (24).

Nucleotide sequence accession number.

The complete nucleotide sequence of pCMY2 was deposited in GenBank under accession no. LC019731.