Emergence of Klebsiella pneumoniae Isolates Producing Inducible DHA-1 β-Lactamase in a University Hospital in Taiwan

Bacterial isolates and patients.The standard screening and confirmation methods for the detection of extended-spectrum β-lactamases (19) were routinely performed at the Department of Pathology, National Cheng Kung University Hospital, a 900-bed teaching hospital in southern Taiwan. Between January 1999 and September 2001, 10 nonrepetitive isolates of K. pneumoniae from 10 patients demonstrated reduced inhibition zone diameters for both ceftazidime and cefotaxime in combination with clavulanic acid versus those for ceftazidime and cefotaxime when tested alone (see Table 1), suggesting production of β-lactamases induced by clavulanic acid. All these isolates were identified by conventional techniques (9) and/or the API 20E system (bioMérieux, Marcy l'Etoile, France). The medical records of the patients from whom the isolates were recovered were reviewed.

Antagonism testing.The disk antagonism method initially used to detect inducibility of chromosomal β-lactamases (16) was performed with a slight modification to test the 10 K. pneumoniae isolates. Disks of inducing agents and disks of cephalosporins were placed on the surface of Mueller-Hinton agar plates and separated by 25 mm (see Fig. 1). The cephalosporins used were cefotaxime, ceftazidime, aztreonam, and cefepime. Clavulanic acid (10 μg) and cefoxitin (30 μg) were used as inducing agents. The plates were examined after overnight incubation at 37°C.

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FIG. 1.

Double-disk antagonism tests with 30 μg of cefoxitin (A) and 10 μg of clavulanic acid (B). ATM, aztreonam; CAZ, ceftazidime; CLA, clavulanic acid; CTX, cefotaxime; FEP, cefepime; FOX, cefoxitin.

Susceptibility testing.MICs were determined by the standard agar dilution method (18). The antimicrobial agents and their sources were as follows: amoxicillin and clavulanic acid, SmithKline Beecham Pharmaceuticals, Surrey, United Kingdom; aztreonam and cefepime, Bristol-Myers Squibb, New Brunswick, N.J.; cefotaxime, Hoechst-Roussel Pharmaceuticals, Inc., Somerville, N.J.; cefoxitin, Sigma Chemical Company, St. Louis, Mo.; ceftazidime, Glaxo Group Research Ltd., Greenford, United Kingdom; and imipenem, Merck Sharp & Dohme, West Point, Pa. The susceptibilities to six non-β-lactam agents were determined by the standard disk diffusion method (19). Antimicrobial disks were obtained from Becton Dickinson Microbiology Systems, Cockeysville, Md., including amikacin, ciprofloxacin, gentamicin, ofloxacin, tobramycin, and trimethoprim-sulfamethoxazole.

IEF.Crude preparations of β-lactamases were obtained from the isolates by sonication (7) and subjected to analytical isoelectric focusing (IEF) as described previously (17, 30). Cells induced by 16 μg of cefoxitin per ml were incubated for 3 h before harvesting (2). β-Lactamase activity was detected by overlaying the gels with 0.5 mM nitrocefin in 0.1 M phosphate buffer, pH 7.0.

PCR and DNA sequencing.Plasmids from the isolates were extracted by a rapid alkaline lysis procedure (27) and used as templates in PCRs. The entire bla_DHA-1 gene was amplified with the oligonucleotide primers DHA-1A (5′-CTGATGAAAAAATCGTTATC-3′) and DHA-1B (5′-ATTCCAGTGCACTCAAAATA-3′), corresponding to nucleotides −3 to 17 and 1138 to 1119, respectively, of the DHA-1 structural gene (2). The PCR conditions were as follows: 3 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C; and finally 7 min at 72°C. The entire sequences of bla_TEM- and bla_SHV-related genes were amplified with the primer pairs as described previously (30). The amplicons were purified with a commercial kit (Roche Molecular Biochemicals, Mannheim, Germany) and sequenced on an ABI Prism 310 sequencer analyzer (Applied Biosystems, Foster City, Calif.).

Transfer of resistance.Conjugation experiments were performed as described previously (24, 30) with streptomycin-resistant Escherichia coli C600 as the recipient (1). Tryptic soy agar plates supplemented with 500 μg of streptomycin (Sigma) per ml and 64 μg of cefoxitin per ml were used to select the transconjugants. K. pneumoniae strain W142 harboring bla_CMY-8 was used as the control (30).

Plasmid analysis and Southern hybridization.Plasmids from the isolates were analyzed by electrophoresis on a 0.8% agarose gel. E. coli strain NCTC 50192 (National Collection of Type Cultures, London, United Kingdom), which contained four plasmids of 7, 36.3, 63.8, and 148.5 kb, was used as a source of molecular size markers. The gel was stained with ethidium bromide (Sigma), visualized under UV light, and subjected to Southern hybridization according to the original protocol (26). The bla_DHA-1-specific probe was a PCR-generated amplicon labeled with [α-³²P]dCTP (Amersham Pharmacia Biotech) by the random priming technique with a commercial kit (Gibco-BRL Life Technologies, Gaithersburg, Md.).

Ribotyping.The chromosomal DNA of the isolates was extracted and purified as described previously (23). The genomic DNA was restricted with EcoRI or BstEII (Roche Molecular Biochemicals) (20). The digests of chromosomal DNA were electrophoresed at 35 V for 18 h in a 0.8% agarose gel, transferred to a nylon membrane (Amersham Pharmacia Biotech), and then hybridized with a [α-³²P]dCTP-labeled cDNA copy of E. coli rRNA (Roche Molecular Biochemicals) obtained by reverse transcription with avian myeloblastosis virus reverse transcriptase (Gibco-BRL) as described previously (23).

Inducibility of β-lactamases.In the standard extended-spectrum β-lactamases confirmatory test, the reduced zone diameters for ceftazidime and cefotaxime in combination with clavulanic acid versus those for ceftazidime and cefotaxime tested alone among the 10 K. pneumoniae isolates ranged from 2 to 10 mm (mean, 5.6 mm) and 3 to 8 mm (mean, 5.6 mm), respectively, suggesting production of β-lactamases induced by clavulanic acid (Table 1). Inducibility of the β-lactamases was further recognized by the disk antagonism test, which demonstrated blunting of the cephalosporin disks adjacent to the cefoxitin and clavulanic acid disks (Fig. 1).

Susceptibility testing.The results of the susceptibility tests are shown in Table 1. All 10 isolates exhibited high-level resistance to amoxicillin-clavulanic acid and cefoxitin. In the presence of clavulanic acid, a one to four twofold concentration increase in the MICs of ceftazidime, cefotaxime, and aztreonam was noted, while the changes after addition of clavulanic acid in the MICs of cefepime and imipenem were not obvious.

Identification of β-lactamases.IEF demonstrated that all 10 isolates displayed three bands of β-lactamase activity with pIs of 5.4, 7.6, and 7.8. The pI 7.6 band probably represented the chromosomal SHV-1 or SHV-11 type β-lactamase of K. pneumoniae (15, 30), the pI 5.4 band might represent the TEM-1 β-lactamase (15), and the pI 7.8 band might represent the β-lactamase responsible for the inducible resistance phenotype.

A 1,141-bp fragment was amplified by PCR with the bla_DHA-1-specific primers for all 10 K. pneumoniae isolates. The amino acid sequences of the PCR products deduced from the sequence analysis were identical to the plasmid-mediated cephalosporinase DHA-1 from S. enterica serovar Enteritidis (2). The DHA-1 cephalosporinase was consistent with the pI 7.8 β-lactamase demonstrated by IEF (2). All the isolates also carried bla_TEM-1 and bla_SHV-11, which were identified by PCR with the bla_TEM- and bla_SHV-specific primers and sequence analysis.

Conjugation experiments and plasmid analysis.Conjugation experiments failed to demonstrate transfer of inducible cephalosporin resistance from any of the isolates. Cefoxitin resistance was transferred from the control strain to E. coli C600 at a frequency of 10⁻³ to 10⁻⁴ per donor cell. Six different profiles were demonstrated by plasmid analysis among the 10 isolates (Fig. 2). In all isolates analyzed, the presence of a plasmid of approximately 70 kb was detected. Southern hybridization with the bla_DHA-1-specific probe showed that bla_DHA-1 was located on the 70-kb plasmid (data not shown).

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FIG. 2.

Plasmid profiles of the 10 K. pneumoniae isolates. Lanes 1 to 10, isolates 387, 416, 1490, 1596, 197, 274, 281, 325, 397, and 1067, respectively; lane M, E. coli NCTC 50192, which contains four plasmids with molecular sizes of 7, 36.2, 63.8, and 148.5 kb.

Ribotyping.The genetic relationship among the 10 K. pneumoniae isolates was investigated by ribotyping with two different endonucleases. Patterns with at least two discordant bands were considered different (20). The results are listed in Table 2 and partially shown in Fig. 3. Both EcoRI and BstEII generated seven different patterns. Isolates 1490 and 1596, both of which were collected in early 2000, and isolates 197, 274, and 281, which were all collected in late 2000, had identical ribotypes, suggesting that they derived from two endemic clones.

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FIG. 3.

Ribotypes generated by EcoRI. Lane 1, molecular size marker kit II (Roche Molecular Biochemicals); lanes 2 to 11, isolates 387, 416, 1490, 1596, 197, 274, 281, 325, 397, and 1067, respectively; lane 12, 1-kb ladder (Promega Co., Madison, Wis.).

Clinical characteristics.Three isolates were recovered from sputum samples and were considered colonizers. The other seven isolates were associated with three urinary tract infections, two wound infections, one intra-abdominal infection, and one bloodstream infection. The clinical characteristics of the patients infected with or colonized by the DHA-1-producing isolates are summarized in Table 2. Six patients had undergone hemodialysis due to either chronic or acute renal failure before isolation. Five isolates were obtained >48 h after the patients were admitted to the hospital. Three of the five isolates were from the patients in the surgery wards, and two isolates were from the patients in the intensive care units. Although the remaining five isolates were obtained within 48 h after the current admission, all patients with these isolates had been hospitalized in the teaching hospital 1 to 5 months earlier. Notably, four of them had been on hemodialysis either in the university hospital or at community hospitals.

All nine patients for whom complete medical records were available had been exposed to β-lactam agents within 2 weeks before isolation of the DHA-1 producers. Patients 3, 4, 6, and 8 received no specific antimicrobial agents for the DHA-1 producers, and patient 7 was not treated for the urinary tract infection at the university hospital. Patients 2 and 9 received ciprofloxacin and trimethoprim-sulfamethoxazole, respectively, for 2 weeks, and the K. pneumoniae strains were not isolated from urine samples afterward. Patient 1 had received cefotaxime and ciprofloxacin, but his deep soft tissue infection was not eradicated until debridement was performed 1 month after admission. Patient 5 received cefotaxime after isolation and died of intra-abdominal hemorrhages and multiorgan failure due to his underlying diseases 1 week later. Whether the bacterial peritonitis was persistent before his death is not clear. Patient 10 was cured of the bloodstream infection with 2 weeks of meropenem and netilmicin therapy. Five patients died during hospitalization, and patient 7 died during the other hospitalization; however, none of the deaths were directly due to infections caused by the DHA-1 producers.