Interspecies Dissemination of the bla Gene Encoding PER-1 Extended-Spectrum β-Lactamase

Abstract

PER-1 extended-spectrum β-lactamase-producing Gram-negative bacilli are resistant to oxyimino-cephalosporins. However, the bla_PER-1 gene has never been reported in Klebsiella pneumoniae. Here, we studied interspecies dissemination of the bla_PER-1 gene by horizontal transfer of Tn1213 among Acinetobacter baumannii, Pseudomonas aeruginosa, and K. pneumoniae. In a K. pneumoniae clinical isolate, the bla_PER-1 gene was located on a 150-kbp incompatibility group A/C plasmid.

The bla gene encoding PER-1 extended-spectrum β-lactamase (ESBL), which can hydrolyze penicillins, oxyimino-cephalosporins, and aztreonam but not oxacillins, cephamycins, and carbapenems, was first detected on a plasmid of Pseudomonas aeruginosa RNL-1 from France in 1991 (9). The widespread dissemination of the gene in Acinetobacter spp. (46%) and P. aeruginosa (11%) in Turkey was reported in 1997, and further dissemination of the gene into European countries, such as Italy, Belgium, and Russia, has been noted (5, 7, 8, 10, 19). In 2003, a high prevalence of the bla_PER-1 gene in Acinetobacter spp. (55%) isolated from patients hospitalized in an intensive care unit (ICU) in Korea was reported, and further dissemination of the gene into Asian countries, such as China, Japan, and India, has also been detected (6, 20-22). The bla_PER-1 gene has been detected mainly in glucose-nonfermenting Gram-negative bacilli, such as P. aeruginosa, Acinetobacter spp., and Alcaligenes faecalis; however, it has also recently been found in Enterobacteriaceae, such as Providencia spp., Proteus spp., Salmonella spp., and Aeromonas media (2, 9, 11-13, 18, 19).

A 68-year-old female who presented with dyspnea and facial paralysis after a bamboo stick injury on the left leg was admitted to the ICU of a tertiary-care hospital in Gwangju, Republic of Korea, on 16 May 2006. She showed symptoms of pneumonia, and Acinetobacter baumannii and Staphylococcus aureus were repeatedly recovered from sputum specimens. Ceftazidime and vancomycin were administered for the treatment of pneumonia; however, she expired on 16 June due to the occurrence of disseminated intravascular coagulation. Klebsiella pneumoniae CS1711 isolate was recovered from the blood specimen obtained 1 day before she died.

Strain CS1711 exhibited resistance to ampicillin, piperacillin, ceftazidime, cefotaxime, cefepime, gentamicin, amikacin, and tetracycline and was susceptible to cefoxitin and imipenem by a disk diffusion assay (5). Synergy was observed between the amoxicillin-clavulanic acid (20 and 10 μg) disk and the ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), and aztreonam (30 μg) disks (Becton Dickinson, Sparks, MD) in double-disk synergy tests, indicating the production of ESBL (17). Agar dilution MIC testing on Mueller-Hinton agar (Difco Laboratories, Detroit, MI) with an inoculum of 10⁴ CFU per spot confirmed MICs of ceftazidime (MIC, 16 μg/ml), cefotaxime (MIC, 64 μg/ml), cefepime (MIC, 64 μg/ml), aztreonam (MIC, 16 μg/ml), cefoxitin (MIC, 4 μg/ml), amikacin (MIC, >256 μg/ml), and ciprofloxacin (MIC, 1 μg/ml) for strain CS1711 (17). Clavulanic acid (Sigma, St. Louis, MO) at a fixed concentration of 4 μg/ml lowered the MICs of ceftazidime, cefotaxime, and cefepime to 1 μg/ml, 0.12 μg/ml, and 0.5 μg/ml, respectively (Table 1).

TABLE 1.

MICs of K. pneumoniae wild strain (CS1711), its transconjugant, and the recipient E. coli J53

Antimicrobial agenta	MIC (μg/ml) of strains
	Wild strain, K. pneumoniae CS1711	Transconjugant, E. coli trcCS1711	Recipient, E. coli J53
Ceftazidime	16	4	0.25
Ceftazidime-clavulanic acid	1	0.12	0.12
Cefotaxime	64	32	0.06
Cefotaxime-clavulanic acid	0.12	0.06	0.06
Cefepime	64	8	0.06
Cefepime-clavulanic acid	0.5	0.06	0.06
Aztreonam	16	8	0.25
Cefoxitin	4	4	4
Amikacin	>256	0.25	0.25
Ciprofloxacin	1	0.25	0.015

^aClavulanic acid was added at a fixed concentration of 4 μg/ml.

The strain transferred an ∼150-kbp plasmid (pCS1711) to the Escherichia coli J53 azide^R recipient in mating experiments in which transconjugants were selected on MacConkey agar (Difco Laboratories, Detroit, MI) plates supplemented with cefotaxime (2 μg/ml) and sodium azide (100 μg/ml) (3). MICs of ceftazidime, cefotaxime, cefepime, amikacin, and ciprofloxacin for the transconjugant (trcCS1711) were 4 μg/ml, 32 μg/ml, 8 μg/ml, 0.25 μg/ml, and 0.025 μg/ml, respectively (Table 1).

PCR and sequencing experiments for the detection of genes encoding TEM-, SHV-, CTX-M-, GES-, VEB-, and PER-type ESBLs were performed as described previously (1) (Table 2). Strain CS1711 carried two β-lactamase genes, bla_PER-1 and bla_CTX-M-9. The location of antimicrobial resistance genes was identified by hybridization of I-CeuI-digested genomic DNA or S1 nuclease-treated linearized plasmids with probes specific for the β-lactamase genes, various replicons of plasmids, and 16S rRNA genes as described previously (16). Clinical isolates of P. aeruginosa (n = 8) and A. baumannii (n = 14), which were recovered from clinical samples of patients hospitalized at the same hospital during May and June 2006, carrying the bla_PER-1 gene were included in this study for comparison. The bla_PER-1 and the bla_CTX-M-9 genes in K. pneumoniae strain CS1711 were located on the ∼150-kbp IncA/C plasmid (pCS1711 in transconjugant E. coli trcCS1711). However, the probe specific for the bla_PER-1 gene did not hybridize with any plasmids in 8 P. aeruginosa isolates and 14 A. baumannii isolates. The probe hybridized with I-CeuI macrorestriction fragments of ∼500 kbp and ∼800 kbp in P. aeruginosa and A. baumannii isolates, respectively. The probe specific for 16S rRNA genes also hybridized with the I-CeuI macrorestriction fragments, indicating chromosomal location of the bla_PER-1 gene in those P. aeruginosa and A. baumannii isolates.

TABLE 2.

Primers used in PCR and sequencing studies for antimicrobial resistance genes

Target gene(s)	Primer name	Sequence (5′ to 3′)	Position in Fig. 1
blaTEM	TEM-F	TCCGCTCATGAGACAATAACC
cluster	TEM-R	ACGCTCAGTGGAACGAAAAC
blaSHV	SHV-F	CGCCGGGTTATTCTTATTTG
cluster	SHV-R	CCACGTTTATGGCGTTACCT
blaVEB	VEB-F	AAAATGCCAGAATAGGAGTAGCA
cluster	VEB-R	TCCACGTTATTTTTGCAATGTC
blaGES	GES-F	CGCTTCATTCACGCACTATT
cluster	GES-R	GTCCGTGCTCAGGATGAGTT
blaCTX-M-1	CTX-M-1F	CCGTCACGCTGTTGTTAGG
cluster	CTX-M-1R	ACGGCTTTCTGCCTTAGGTT
blaCTX-M-9	CTX-M9-F	CAAAGAGAGTGCAACGGATG
cluster	CTX-M9-R	CCTTCGGCGATGATTCTC
blaPER-1	PER-F	CCTGACGATCTGGAACCTTT	1
cluster	PER-R	TGGTCCTGTGGTGGTTTC	2
ISPa12 gene	ISPa12-F	AAGCCCTGTTTTCAGAGCAA	3
	ISPa12-R	AATCAACGTTTCGGCTATCG	4
	ISPa12-mF	GCCGATGCAGGTTATTTTTC	5
	ISPa12-wR	TCATGATTCATATGTGATTTCCAA	6
ISPa13 gene	ISPa13-F	TTTTCAGCAGCAGAGCTTGA	7
	ISPa13-R	CGTTGATTAGCCAGCGTTTT	8
	ISPa13-mF	TGATAAAGAGGCGGGTGAAG	9
	ISPa13-wR	TTTACGCCTCATAGGTATGATCTTTAG	10
gst gene	GST-F	CCCCTTTTGTTCGTCGTTTA	11
	GST-R	AAGGAGTCTGTGCAGGCATT	12

To investigate genetic environments surrounding the bla_PER-1 gene, sequencing experiments of several overlapping PCR fragments obtained from whole DNA of the K. pneumoniae, A. baumannii, and P. aeruginosa isolates with primers corresponding to internal region of Tn1213 were performed as previously described (15). Identically to the results for the bla_PER-1 gene located on the chromosome in P. aeruginosa RNL-1 (GenBank accession no. AY779042), ISPa12 and ISPa13 elements were present upstream and downstream of the bla_PER-1 gene, respectively, in all the isolates studied. The bla_PER-1 gene located on a plasmid in two Salmonella enterica serovar Typhimurium isolates and in strain A. baumannii C.A. has been reported to be preceded by ISPa12 but not followed by ISPa13, while the bla_PER-1 gene located on the plasmid pCS1711 was surrounded by both ISPa12 and ISPa13 elements (4, 14, 15) (Fig. 1). Our results suggest that the bla_PER-1 gene in K. pneumoniae strain CS1711 might be mobilized from bla_PER-1 gene-carrying A. baumannii or P. aeruginosa, since the genetic environments of the gene in those strains were identical.

FIG. 1.

Genetic environment of the bla_PER-1 gene in K. pneumonia CS1711. Numbered arrows indicate positions and directions of the primers used in this study as listed in Table 2.

This report shows further dissemination of the bla_PER-1 gene into Enterobacteriaceae and is the first report of K. pneumoniae carrying the bla_PER-1 gene.