First Identification of IMP-1 Metallo-β-Lactamase in Delftia tsuruhatensis Strain CRS1243 Isolated From a Clinical Specimen

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Delftia tsuruhatensis is a gram-negative bacillus that was first isolated from activated sludge collected from a domestic wastewater treatment plant in Tsuruhata, Japan [1]. The bacteria are motile, slightly curved, and short rods. The species is closely related to and often misidentified as Delftia acidovorans (formerly Comamonas acidovorans) in biochemical tests because of their shared characteristics [1, 2]. However, the original D. tsuruhatensis isolate cannot utilize D-mannitol, whereas most D. acidovorans strains can [3]. We present the first report of an IMP-1 metallo-β-lactamase (MBL)-producing D. tsuruhatensis strain that was isolated from a clinical specimen. The Institutional Review Board of the CHA Bundang Medical Center, Seongnam, Korea, approved this study (approval number: 2020-04-011-001) and waived the need for informed consent.

D. tsuruhatensis is typically susceptible to carbapenem [2, 4]; however, we isolated a carbapenem-resistant strain from a 65-yr-old man diagnosed as having stomach cancer who underwent total gastrectomy. On post-operative day 16, bacterial culture was performed because of onset of fever. A Delftia species (strain CRS1243) was isolated from the surgical drainage fluid. The isolate was identified as D. acidovorans with 99% probability using Vitek 2 GN cards (bioMerieux, Marcy-l’Etoile, France) and with a score of 2.40 using the MALDI Biotyper (Bruker Daltonics, Billerica, MA, USA). However, as the isolated bacteria were unable to metabolize D-mannitol, we performed 16S rRNA gene sequencing and identified the strain as D. tsuruhatensis (100% identity with GenBank accession number HQ731453.1).

The isolate was suspected to produce carbapenemases based on routine antimicrobial susceptibility testing using Vitek AST N212 cards (bioMerieux). Antimicrobial susceptibility was determined according to the CLSI Minimum Inhibitory Concentration (MIC) Interpretive Standards for other non-Enterobacteriaceae using the broth microdilution method [5, 6]. The isolate was not susceptible to amikacin (MIC > 64 μg/mL), levofloxacin (8 μg/mL), ceftazidime (>16 μg/mL), cefepime (>32 μg/mL), ceftriaxone (>32 μg/mL), and meropenem (16 μg/mL), but it was susceptible to piperacillin-tazobactam (16 μg/mL) and minocycline (≤0.5 μg/mL). The modified Hodge test result was positive, and in carbapenemase inhibition testing (Rosco Diag-nostica A/S, Taastrup, Denmark), we observed enlarged inhibition zones when using disks containing meropenem supplemented with dipicolinic acid, indicating production of MBL. An expanded inhibition zone was also detected with disks containing meropenem supplemented with cloxacillin, but not with those containing boronic acid. Moreover, the cefoxitin-Hodge test result was negative. Based on these results, we concluded that this isolate was negative for AmpC β-lactamase activity.

Multiplex PCR was performed to detect the specific MBL. The specific primer pairs for the detection of the New Delhi MBL and Sao Paulo MBL were designed in this study and those for the detection of Verona integron-encoded MBL, imipenemase (IMP), Seoul IMP, and German IMP were selected from a previous report (Table 1). The sequence of the bacterial integron carrying the MBL-encoding gene was identified using primer walking (Table 1). We found that D. tsuruhatensis CRS1243 harbored a gene encoding bla_IMP-1 within a class 1 integron located on a Tn402-like transposon. Between the 5'-conserved segment and the tni module, the gene cassettes included orfE, aac(6')-31-like, orfE, orfE, aac(6')-II, aacA7, bla_IMP-1, aacA7, aac(6')-II, and qacE2 (Fig. 1). The nucleotide sequence of the class 1 integron has been deposited in GenBank under accession number KC170993. While a class 3 integron has been previously identified in a D. tsuruhatensis strain, the gene cassettes were not evaluated functionally [12]. To determine the location of the class 1 integron, we performed plasmid extraction and conjugation [13]. However, we could not identify the band corresponding to the plasmid DNA, and the carbapenem resistance was not transferred to sodium azide-resistant Escherichia coli J53.

Table 1.

Primers used for the PCR analysis of metallo-β-lactamase genes and primer walking

Primer	Sequence (5´ → 3´)	References
NDM-F1	GCC CAA TAT TAT GCA CCC GG	This study
NDM-R	CGG AAT GGC TCA TCA CGA TC	This study
SPM-NF	TGC GGG AGC GCC ATT GTC TG	This study
SPM-NR	TTC CAC CCG TGC CGT CCA AA	This study
VIM-F	GAT GGT GTT TGG TCG CAT A	7
VIM-R	CGA ATG CGC AGC ACC AG	7
IMP-F	GGA ATA GAG TGG CTT AAY TCT C	7
IMP-R	CCA AAC YAC TAS GTT ATC T	7
SIM-F	TAC AAG GGA TTC GGC ATC G	7
SIM-R	TAA TGG CCT GTT CCC ATG TG	7
GIM-F	TCG ACA CAC CTT GGT CTG AA	7
GIM-R	AAC TTC CAA CTT TGC CAT GC	7
INT1-5CS	GGC ATC CAA GCA GCA AGC	8
intl1-1F	ACA TGC GTG TAA ATC ATC GTC G	9
attI-R	CTT TGT TTT AGG GCG ACT GC	This study
aac6-F1	GCT CGT TGA GAT GCT GTT CA	This study
aacA7-R	GAA GCA GCG TAC TTG AGC AA	This study
IMP-1R	CCT TTA ACC GCC TGC TCT AAT G	10
IMP14	AGG CGT GCT GCT GCA ACG ACT TGT	11
qacEd1-R	TGA GCC CCA TAC CTA CAA AGC	9
qacE2-R	ATT TGA GTG TCA GCG ACA GG	This study
TniR-1	GTG TTC GGT ATT TTT GCC GC	This study
TniR-2	GTA ATC CCG AGT TCT TCG CA	This study
TniQ-1	TGT GGT TTC GAC TTC TTC GC	This study
TniQ-2	GAC CAG AAT AGC TTT GCC TG	This study
TniQ-1M	TGT GGT TTC GAC TGC TAC GC	This study
TniB-1	GGA AAT GGA GCA ACT GGC T	This study
TniB-2	TTT CCA ACT GGT CAT CGG AG	This study
TniB-1M	AGA AAT GGA ACA ACT GGC G	This study
TniA-1	TAG AGC GCT GGC TCA CAT T	This study
TniA-2	GGA TGT GGT CGA TGA CAA AG	This study

Fig. 1.

Schematic representation of the 8,846-bp partial DNA sequence of the class 1 integron containing bla_IMP-1 within a Tn402-like module in Delftia tsuruhatensis CRS1243.

Cases of human infection with D. tsuruhatensis are rare [2]. In 2011, D. tsuruhatensis was identified as the etiologic agent of a human catheter-related infection; since then, it has also been associated with other human infections [2]. Recently, Fenollar, et al. [4] identified D. tsuruhatensis as an emerging opportunistic pathogen that should be considered as a cause of infection in patients with underlying disease and those using intravascular devices. The D. tsuruhatensis strain identified in this study was isolated twice in five days from pure cultures from surgical drainage fluid. Treatment with ciprofloxacin, an empirical antibiotic was started; however, piperacillin-tazobactam was administered after confirmation of the antibiotic susceptibility results. The patient’s fever subsided one day after initiation of the treatment.

To the best of our knowledge, this is the first report of IMP-1 MBL production from a D. tsuruhatensis strain. Our findings suggest that clinical microbiologists need to be aware of D. tsuruhatensis as a potential cause of opportunistic infections. We note that the bla_IMP-1 gene linked to a mobile element might spread among Delftia or other bacterial species.