Wide Dissemination of Pseudomonas aeruginosa Producing β-Lactamase bla_KPC-2 Gene in Colombia

Abstract

Ten bla_KPC-2-harboring Pseudomonas aeruginosa isolates from hospitals located in five different Colombian cities have been characterized. Isolates were multidrug resistant, belonged to five different pulsotypes, and possessed naturally chromosome-encoded bla_AmpC and bla_OXA-50 genes and the acquired bla_KPC-2 gene. In most cases, the bla_KPC-2 genes were carried by plasmids of different sizes and were associated with Tn4401b or a new structure containing only part of the Tn4401 sequence. This study revealed that several clones of P. aeruginosa producing bla_KPC-2 are disseminating in Colombia.

TEXT

Carbapenem resistance in Pseudomonas aeruginosa isolates represents a major threat to the management of nosocomial infections and involves several mechanisms, such as porin modifications, efflux pump overexpression, and acquired carbapenem-hydrolyzing β-lactamases (carbapenemases) (10, 14). Carbapenemases identified in P. aeruginosa are mostly metallo-ß-lactamases (14), but the Ambler class A β-lactamase KPC has also been reported (17). KPC carbapenemases were described initially in a report of a Klebsiella pneumoniae isolate collected in 2001 in North Carolina (20) and have since rapidly emerged and disseminated in the world, in enterobacterial species in particular (4, 12). KPC-producing P. aeruginosa isolates were reported first in 2006 from Colombia (17) and subsequently in Puerto Rico (19), in Trinidad and Tobago (1), in the southern part of the United States (13), and very recently in China (6). The rapid spread of KPC enzymes in Enterobacteriaceae has been linked to the genetic elements carrying the bla_KPC-2 gene: plasmids of different sizes harboring a Tn3-like transposon, Tn4401 (4, 11). Nevertheless, other genetic structures have been characterized with different insertion se-quences (IS) upstream of the bla_KPC gene, as seen in a P. aeruginosa isolate harboring bla_KPC-5 (8, 19). Until now, little has been known about the clonal relationships and genetic background of KPC-producing P. aeruginosa strains. The aim of the present work was to characterize 10 bla_KPC-2-harboring P. aeruginosa isolates collected from Colombian hospitals located in different cities scattered throughout the country and sent to the International Center for Medical Research and Training (CIDEIM), Cali, Colombia, between 2006 and 2010.

Molecular typing by pulsed-field gel electrophoresis (PFGE) identified five pulsotypes (A to E) among the isolates (Table 1; Fig. 1). Two closely related patterns (A1 and A2), differing by only one band, were identified in isolates from three different wards in the same hospital in Medellin, Colombia. Four isolates belonged to clone A1, and two others belonged to clone A2. The other isolates were unrelated, differing by at least seven bands, thus suggesting heterogeneity among KPC-producing P. aeruginosa strains from Colombia. Four distinct allelic profiles were obtained by multilocus sequence type (MLST) analysis (Table 1) (3): ST308 (isolates PA-1 and PA-4 to -8), ST235 (isolates PA-9 and PA-10), ST1006 (isolate PA-2), and ST1060 (isolate PA-3). These results matched perfectly with the PFGE results. The four sequence types identified were not single- or double-locus variants of each other. Among them, only ST235 had already been identified as a single-locus variant of ST227 and ST230; all three correspond to clonal complex CC11 (7), which has been isolated in several countries and has been shown to be associated with various β-lactamases (5, 7). The dissemination of bla_KPC into genetically unrelated P. aeruginosa isolates has already been described in Puerto Rico (19), where seven different clones have been found, highlighting the rapid spread of the bla_KPC gene in this area.

Table 1.

Origin, structure of Tn4401, other β-lactamases, plasmid analysis, pulsotype, and sequence type of the P. aeruginosa isolates

Isolate	Origin in Columbia	Yr of isolation	Site of isolation	Location	PCR and sequencing result (Tn4401 homology)				blaKPC-2-positive plasmid				Bacterial isolate characteristic
									Name	Size (kb)	Transformant		Pulsotype	Sequence type
					blaKPC-2	TnpA	ISKPN6	ISKPN7			ECa	PAb
PA-1	Medellin	2006	Bronchial secretion	Tertiary care center	KPC-2	+	+	+	pCOL	45	+	+	A1	ST308
PA-2	Bogota	2006	Stool	Unknown	KPC-2	−	+	−	pPA-2	13	+	+	B	ST1006
PA-3	Barranquiila	2006	Bronchial secretion	Intensive care unit	KPC-2	+	+	+	pPA-3	60	−	−	C	ST1060
PA-4	Medellin	2006	Bone	General wardc	KPC-2	+	+	+	pPA-4	45	+	+	A2	ST308
PA-5	Medellin	2006	Surgical wound	General ward	KPC-2	+	+	+	pPA-5	45	+	+	A2	ST308
PA-6	Medellin	2006	Surgical wound	General ward	KPC-2	+	+	+	pPA-6	45	+	+	A1	ST308
PA-7	Medellin	2006	Urine	General ward	KPC-2	+	+	+	pPA-7	45	+	+	A1	ST308
PA-8	Medellin	2006	Tracheal aspirate	Intensive care unit	KPC-2	+	+	+	pPA-8	45	+	+	A1	ST308
PA-9	Cali	2007	Peritoneal fluid	General ward	KPC-2	+	+	+	−	−	−	−	D	ST235
PA-10	Pereira	2010	Urine	Intensive care unit	KPC-2	+	+	+	−	−	−	−	E	ST235

^aTransformants into E. coli (EC) DH10B.

^bTransformants into P. aeruginosa (PA) KG2505.

^cGeneral ward: patients hospitalized for surgical or medical disease.

Fig. 1.

Dendrogram of pulsed-field gel electrophoresis data (SpeI digests) showing genetic relatedness between the 10 KPC-producing reference isolates. M, molecular weight.

Antibiotic susceptibility was determined by the disk diffusion method, and MICs of carbapenems were determined using Etest strips (bioMérieux, Marcy l'Etoile, France) and interpreted according to Clinical and Laboratory Standards Institute guidelines (2). The 10 isolates were resistant to all penicillins and expanded-spectrum cephalosporins tested and showed carbapenem MIC values above 32 mg/ml (Table 1). The use of cloxacillin (200 mg/liter)-containing Mueller-Hinton (MH) agar plates did not alter the antibiogram results. All isolates remained susceptible to colistin. Eight isolates (PA-1 and PA-4 to -10) were also fully resistant to all tested aminoglycosides (amikacin, gentamicin, netilmicin, and tobramycin) and to ciprofloxacin; one isolate (PA-2) was susceptible to all tested aminoglycosides and resistant to ciprofloxacin; and a single isolate (PA-3) was susceptible to ciprofloxacin, amikacin, and fosfomycin, according to guidelines from CA-SFM (http://www.sfm-microbiologie.org).

PCR and sequencing (Table 2) (11) revealed that all isolates possessed bla_KPC-2 genes and naturally and chromosomally encoded bla_AmpC and bla_OXA-50 genes, but bla_SHV, bla_TEM, bla_CTX-M, bla_VIM, bla_IMP, and bla_OXA-9 genes could not be evidenced.

Table 2.

Primers used in this study

Name of primer	Fig. 2 lane no.	Sequence (5′-3′)	Reference
KPC-A	1	CTGTCTTGTCTCTCATGGCC	11
KPC-B	2	CCTCGCTGTGCTTGTCATCC	11
3098U	3	TGACCCTGAGCGGCGAAAGC	11
4914L	4	GAAGATGCCAAGGTCAATGC	11
TnpA-178U	5	CTTGCAGATCGGCTACTTCA	This study
TnpA-1198U	6	CGCCATCTTCGTCCACTGTA	This study
TnpA-2537L	7	GCCCTGCGTCATTTCCTTCA	This study
ISKpn7-24U	8	CCCACCCCAGCGAAGTAAAAC	This study
ISKpn7-1916L	9	GACCCACTTTACCCCTGAAT	This study
ISKpn6-82U	10	CGTCGGCGCCAAGGATACCA	This study
ISKpn6-1429L	11	ATCTGCTGCCCCCTTCTCTG	This study
141R-6	12	TCACCGGCCCTCACCTTTGG	11
EcoRI-out	13	CACCCGACCTGGACGAACTA	11
Delta SHVa		AAGATCCACTATCGCCAGCAG	4
Delta SHVb		ATTCAGTTCCGTTTCCCAGCGG	4
Pre-TEM1		GTATCCGCTCATGAGACAATA	4
Pre-TEM2		TCTAAAGTATATATGAGTAAACTTGGTCTG	4
CTX-MA		CGCTTTGCGATGTGCAG	4
CTX-MB		ACCGCGATATCGTTGGT	4
VIM-B		ATGGTGTTTGGTCGCATATC	11
VIM-F		TGGGCCATTCAGCCAGATC	1
IMP-2004A		ACAYGGYTTGGTDGTTCTTG	1
IMP-2004B		GGTTTAAYAAAACAACCACC	1
OXA-9A		TTCGTTTCCGCCACTCTCCC	4
OXA-9B		ACGAGAATATCCTCTCGTGC	4

By the Kieser extraction method (9), the six isolates from Medellin (PA-1 and PA-4 to -8) and Barranquilla (PA-3) showed several plasmids of different sizes (45 kb to ≥150 kb) (Fig. 2A). One isolate from Bogota (PA-2) showed only one plasmid (13 kb), and no plasmid could be evidenced for strains from Cali (PA-9) and Pereira (PA-10), suggesting a chromosomal location of the bla_KPC-2 gene, as has already been described in P. aeruginosa isolates (17) (Fig. 2B). Only one plasmid hybridized with an internal probe for a bla_KPC gene in each isolate, with sizes ranging from 13 to 60 kb (Fig. 2A; Table 1). Transformants into Escherichia coli DH10B could be obtained with all the natural plasmids except with the 60-kb pPA-3 plasmid. No transformants could be obtained with Kieser extracts from PA-9 and PA-10. No other antibiotic resistance marker was cotransferred. The natural plasmids pPA-1, pPA-2, and pPA-4 to -8 were also electroporated into the AmpC-deficient P. aeruginosa KG2505 strain (16) and conferred a high level of resistance to all ß-lactams tested, including carbapenems. Previous studies have reported that OprD porin was absent in numerous KPC-producing P. aeruginosa isolates (17, 18) and is associated both with overexpression of the mexAB-oprM efflux pump and with increases in carbapenem MICs. However, in the present study, electroporation of plasmids harboring the bla_KPC-2 gene was sufficient to confer a high level of resistance to the naturally susceptible P. aeruginosa KG2505 isolate.

Fig. 2.

(A) Plasmid extractions from cultures of the different isolates by the Kieser method. (B) Southern hybridization carried out with an internal probe for the bla_KPC-2 gene. Lane 1, P. aeruginosa PA-1; lane 2, P. aeruginosa PA-2; lane 3, P. aeruginosa PA-3; lane 4, P. aeruginosa PA-4; lane 5, P. aeruginosa PA-5; lane 6, P. aeruginosa PA-6; lane 7, P. aeruginosa PA-7; lane 8, P. aeruginosa PA-8; lane 9, P. aeruginosa PA-9; lane 10, P. aeruginosa PA-10; lane 11, E. coli 50192 harboring four (7-, 48-, 66-, and 154-kb) plasmids. C, chromosome.

A PCR mapping approach revealed that the bla_KPC-2 gene was associated with Tn4401b isoform in PA-1 and PA-3 to -10 (Table 2, Fig. 3). The natural plasmids pPA-1 and pPA-4 to -8 were extracted from electroporants and directly sequenced using outward-directed primers located next to the inverted repeats (IRs). For these six plasmids, the flanking sequences of Tn4401b were identical; it was inserted into an open reading frame (ORF) of 297 bp initially described as being carried on plasmid pRSB105, which was extracted from uncultured bacteria of a sewage plant in Germany (15). Upon insertion, Tn4401b generated identical 5-bp GCGCT target site duplications. For plasmid pPA-2, PCR mapping gave negative results, suggesting a different genetic organization, which was confirmed by direct sequencing of the plasmid. Only a 2,072-bp region, including the bla_KPC-2 gene and an ISKpn6-like ORF previously described in a study of K. pneumoniae isolates collected in China, was identical to Tn4401 (8). This ISKpn6-like element shared a 1,039-bp fragment with ISKpn6. The target site duplication and the left inverted repeat (IRL) as described in Tn4401 were present, but the ORF encoding the putative transposase (439 amino acids in ISKpn6) was truncated in its N terminus by a 117-bp fragment encoding the C-terminal region of a protein sharing 100% amino acid identity with the traN protein from plasmid pFBAOT6 previously described in a study of Aeromonas punctata (GenBank accession no. NC_006143) (14a). Upstream of the bla_KPC-2 gene, only a 73-bp segment, which is interrupted by a truncated bla_TEM gene, is identical to Tn4401.

Fig. 3.

Schematic representations of genetic structures surrounding the bla_KPC gene in different P. aeruginosa isolates. (A) P. aeruginosa PR280 harboring bla_KPC-5 from Puerto Rico (19); upstream of the bla_KPC-5 gene, a portion of the transposable element Tn5563, previously described in plasmid pRA2 from P. alcaligenes, was found. (B) Novel structure characterized in PA-2 from Colombia. (C) Structure of Tn4401b. The dotted rectangles indicate the common regions between the different structures. Horizontal arrows indicate genes and their corresponding transcription orientations. Gray triangles represent the left inverted repeat (IRL) and right inverted repeat (IRR) of Tn4401. Small and empty triangles represent the inverted repeats of ISKpn6 and ISKpn7. Target site duplications (TSDs) are indicated above the sequence. Small black triangles above the sequence represent the primers listed in Table 2.

Unlike Enterobacteriaceae, P. aeruginosa isolates expressing KPC carbapenemase seem to be geographically limited. The presence of bla_KPC in P. aeruginosa isolates is worrying in a species that is known to be prone to becoming carbapenem resistant by multiple mechanisms. Our analysis of several KPC-producing P. aeruginosa isolates from different Colombian hospitals revealed the spread of different clones that harbor different plasmids and different genetic structures, including the Tn4401b isoform associated with the bla_KPC-2 gene. The emergence of bla_KPC-2 in different species of Enterobactriaceae and the spread of this gene to P. aeruginosa isolates in different countries emphasizes the potential for dissemination worldwide. Since phenotypic detection is still difficult, it remains to be determined to what extent KPC-producing P. aeruginosa have spread worldwide, particularly in South America.

Nucleotide sequence accession number.

The nucleotide sequence reported in this paper has been assigned to the GenBank nucleotide database (accession number JN545009).