Characterization of Piperacillin/Tazobactam-Resistant Klebsiella oxytoca Recovered from a Nosocomial Outbreak

We characterized 12 clinical isolates of Klebsiella oxytoca with the extended-spectrum β-lactamase (ESBL) phenotype (high minimum inhibitory concentration [MIC] values of ceftriaxone) recovered over 9 months at a university hospital in Japan. To determine the clonality of the isolates, we used pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and PCR analyses to detect bla RBI, which encodes the β-lactamase RbiA, OXY-2-4 with overproduce-type promoter. Moreover, we performed the isoelectric focusing (IEF) of β-lactamases, and the determination of the MICs of β-lactams including piperacillin/tazobactam for 12 clinical isolates and E. coli HB101 with pKOB23, which contains bla RBI, by the agar dilution method. Finally, we performed the initial screening and phenotypic confirmatory tests for ESBLs. Each of the 12 clinical isolates had an identical PFGE pulsotype and MLST sequence type (ST9). All 12 clinical isolates harbored identical bla RBI. The IEF revealed that the clinical isolate produced only one β-lactamase. E. coli HB101 (pKOB23) and all 12 isolates demonstrated equally resistance to piperacillin/tazobactam (MICs, >128 μg/ml). The phenotypic confirmatory test after the initial screening test for ESBLs can discriminate β-lactamase RbiA-producing K. oxytoca from β-lactamase CTX-M-producing K. oxytoca. Twelve clinical isolates of K. oxytoca, which were recovered from an outbreak at one university hospital, had identical genotypes and produced β-lactamase RbiA that conferred resistance to piperacillin/tazobactam. In order to detect K. oxytoca isolates that produce RbiA to promote research concerning β-lactamase RbiA-producing K. oxytoca, the phenotypic confirmatory test after the initial screening test for ESBLs would be useful.

We experienced an outbreak caused by K. oxytoca with the ESBL phenotype (high minimum inhibitory concentration [MIC] value of ceftriaxone [CRO]) at a university hospital in Japan. Here, we report the characterization of clinical isolates of K. oxytoca derived from this outbreak over a period of 9 months.

Ethics statement
We used clinical information concerning clinical isolates analyzed in this study. All the clinical information was approved by the ethical committee of the Aichi Medical University Graduate School of Medicine.

Clinical information
This outbreak was declared in June 2009 and containment of the outbreak was declared in December 2010. The outbreak has been ended by enforcing strict hand hygiene, strict contact precaution and promotion of antimicrobial stewardship. K. oxytoca clinical isolates NUBL-1520NUBL- , 1521NUBL- , 1522NUBL- , 1523NUBL- , 1524NUBL- , 1525NUBL- , 1526NUBL- , 1527NUBL- , 1528NUBL- , 1529NUBL- , 1530, and 1531 were recovered from 8 different patients at one university hospital in Aichi, Japan from 2009 June to 2010 February (Table 1). All the patients were inpatients, admitted at the identical ward of neurosurgery, for various operations. The outcomes of all the patients were survival or change of hospital.

Reagents
Ampicillin (AMP) and cefotaxime (CTX) were purchased from Wako Pure Chemical Industries, LTD. Piperacillin (PIP) and tazobactam were purchased from LKT Laboratories, Inc. Imipenem (IPM) was purchased from Ark Pharm. The disks used for Screening and Confirmatory Tests for ESBLs contained the antibiotics as follows: cefpodoxime (CPD), ATM, CRO, ceftazidime (CAZ), and CTX disks were purchased from Becton, Dickinson and Company. Clavulanic acid (CLA) was purchased from Wako Pure Chemical Industries, LTD.

Pulsed-field gel electrophoresis (PFGE)
Plugs were prepared using suspensions of clinical isolates with an optical density of 0.8; these plugs had treated with 2 mg/ml of lysozyme solution at 37°C for 6 h and 1 mg/ml of proteinase K solution at 55°C for 8 h. The digested plugs were incubated with XbaI (Takara). We performed PFGE for 24 h using a CHEF-DR III System (BioRad). Gels were stained with 0.5 μg/ ml of ethidium bromide for 1 h.

Multi-locus sequence typing (MLST)
We performed MLST analysis of the K. oxytoca isolates as described previously [23]. We isolated chromosomal DNA using a Wizard Genomic DNA Purification Kit (Promega). The seven housekeeping genes were amplified using PCR with the high-fidelity PrimeSTAR HS . We performed PCR reaction using the purified chromosomal DNA as templates, high fidelity DNA polymerase, PrimeSTAR HS DNA polymerase (Takara), and previously described primers, OXY-383 and OXY-S [7]. The nucleotide sequences of the amplicons were determined as described above.

Isoelectric focusing (IEF) of β-lactamases
To extract β-lactamases from the clinical isolate NUBL-1520, we performed a freeze-thaw procedure [24] and subjected the resulting supernatant to IEF using an Invitrogen system. IEF was conducted for 1 h at 100 V, 2 h at 200 V and 30 min at 500 V. The β-lactamase in the gel was detected using 0.05% nitrocefin solution [25].

Determination of MICs
The MICs of AMP, PIP, TZP, CTX, and IPM were determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) using the agar dilution method [26]. E. coli ATCC 25922 and E. coli ATCC 35218 strains served as controls.

Screening and Confirmatory Tests for ESBLs
We performed the disk diffusion method recommended by CLSI called the Screening and Confirmatory Tests for ESBLs [26]. In the Initial Screen Test, we used CPD, and CRO, and ATM disks. For K. oxytoca, the breakpoints of the CPD zone, CRO zone, and ATM zone are 17 mm, 25 mm, and 27 mm, respectively. According to the CLSI, "Zones above may indicate ESBL production." In Phenotypic Confirmatory Test, we used CAZ, CAZ-CLA, CTX, and CTX-CLA disks. Confirmatory testing requires the use of both CAZ and CTX, alone and in combination with CLA. According to the CLSI, "a 5 mm increase in the zone diameter for either antimicrobial agent tested in combination with CLA vs. its zone when tested alone = ESBL." We used NUBL-793 and 810, which have been already confirmed as β-lactamase CTX-M-producing K. oxytoca.

MICs at a clinical setting
The MICs for the 12 clinical isolates of K. oxytoca determined at a microbiological laboratory of a university hospital are shown in Table 2. All isolates were resistant to cefazolin, cefotiam, and CRO. At first, the laboratory technicians missed the high MIC values of sultamicillin and cefoperazone/sulbactam. Therefore, they suspected these clinical isolates as ESBL-producing K. oxytoca, because of their high MIC values of CRO.

PFGE analysis
All clinical isolates exhibited the identical pulsotype (Fig 1), suggesting that they possessed identical genotypes, which indicates that the outbreak was caused by the same clinical isolate.

MLST
All 12 clinical isolates were ST9, indicating that they possessed the identical genotype.

PCR detection of the β-lactamase RbiA gene
Because of the high MIC values of sultamicillin and cefoperazone/sulbactam and our previous findings that the β-lactamase RbiA confers resistance to β-lactamase inhibitors upon K. oxytoca [20], we performed PCR and nucleotide sequence analyses to detect bla RBI and found the bla RBI sequences of all isolates were identical (Accession Number D84548), including the -35 and -10 regions, the Shine-Dalgarno sequence, and the coding region. This supports the clonal origin of the 12 clinical isolates.

IEF analysis of β-lactamases
To determine the number of β-lactamases produced by the clinical isolates, we performed IEF (Fig 2). A single band was detected at pH 5.6, suggesting that NUBL1520 produces 'only one' β-lactamase and supporting that only one β-lactamase produced by NUBL1520 is β-lactamase RbiA [20,27].

Determination of MICs
Although the wide use of TZP started recently in Japan and there are a few reports concerning TZP resistant K. oxytoca that produce OXY-2 type β-lactamase [12,28], it remained to be determined whether K. oxytoca strains that produce RbiA are resistant to TZP. Therefore, we

Initial Screening and Confirmatory Tests for ESBLs
Although it was previously reported that many β-lactamase K1-overproducing K. oxytoca strains show false-positive in ESBL tests [29], no data were available indicating whether the initial screening and confirmatory tests for ESBLs recommended by CLSI detect K. oxytoca clinical isolates that produce RbiA. Therefore, we tested the clinical isolates along with the control strains K. oxytoca NUBL793 and NUBL810 that produce CTX-M. In the initial screening test, the diameters of inhibition surrounding the CPD, ATM, and CRO disks in plates containing NUBL793, NUBL810 as well as those of all clinical isolates were less than the cut-off values recommended by CLSI, suggesting that all clinical isolates may produce ESBLs (Table 4). In the phenotypic confirmatory test, NUBL793 and NUBL810 showed an obvious increase (5 mm)  of the diameters of the CTX-CLA and CTX disks; however, none of the 12 clinical isolates showed the necessary increase of the diameters between CTX-CLA disk and CTX alone disk, and CAZ-CLA disk and CAZ alone disk ( Table 5), suggesting that the phenotypic confirmatory test discriminates K. oxytoca strains that produce RbiA from those that produce CTX-M.

Discussion
We show here that 12 clinical isolates of K. oxytoca, which we recovered from an outbreak at one university hospital, had identical genotypes and produced β-lactamase RbiA that conferred resistance to TZP. Moreover, we demonstrated that the phenotypic confirmatory test after the initial screening test for ESBLs recommended by CLSI is useful for discriminating K. oxytoca clinical isolates that produce RbiA from those that produce CTX-M. It has been reported previously that some ESBL-producing clinical isolates of Klebsiella spp. are resistant to TZP [30,31], and that only 12 of 25 Enterobacteriaceae strains producing ESBLs are susceptible to TZP [32]. However, although several studies have examined ESBL-producing clinical isolates [33], the number of reports concerning K. oxytoca clinical isolates producing β-lactamase RbiA is limited. Therefore, in order to promote research on K. oxytoca clinical isolates producing β-lactamase RbiA, it is important to discriminate K. oxytoca clinical isolates that produce RbiA from ESBL-producing K. oxytoca clinical isolates and to characterize K. oxytoca clinical isolates producing β-lactamase RbiA. TZP is often prescribed for patients treated in the hospital studied here (data not shown). It is possible that the outbreak described here was caused by selection by TZP of K. oxytoca strains that produce RbiA. Moreover, the amount of TZP prescribed in Japan may be increasing in concert with the increase in ESBL-producing Enterobacteriaceae. Therefore, it is reasonable to assume that outbreaks similar to that described here will occur again.
It is difficult to readily discriminate between ESBL-producing K. oxytoca strains and those that produce RbiA because of the ESBL-phenotype (high MIC values of CRO et al.) of the latter. However, we show here that K. oxytoca clinical isolates that produce RbiA are resistant to TZP. Moreover, in our hands, the confirmatory test after the initial screening test for ESBLs recommended by CLSI were useful for discriminating between the two K. oxytoca phenotypes. In order to detect K. oxytoca isolates that produce RbiA to promote research concerning β-lactamase RbiA-producing K. oxytoca, the phenotypic confirmatory test after the initial screening test for ESBLs would be useful. Confirmatory testing requires the use of both CAZ and CTX, alone and in combination with CLA. According to the CLSI, "a 5 mm increase in the zone diameter for either antimicrobial agent tested in combination with CLA vs. its zone when tested alone = ESBL." Abbreviations: ESBL, extended-spectrum β-lactamase; CAZ, ceftazidime; CLA, clavulanic acid; CTX, cefotaxime.