First Description of KPC-2-Producing Pseudomonas putida in Brazil

Abstract

This work reports the identification of the first case of a KΡC-2-producing Pseudomonas putida isolate (PP36) in Brazil. The PP36 isolate was resistant to all the antimicrobials tested except polymyxin B. In addition to the discovered bla_KPC-2 gene, genetic analysis showed the presence of a class 1 integron containing the dhfrXVb gene and the new allele arr-6, which codes for resistance to rifampin. These elements were found in an IncFI 65-kb plasmid.

TEXT

Since the first detection of a Klebsiella pneumoniae isolate harboring the bla_KPC gene in a large plasmid in 1996 in North Carolina (15) and until 2005, the geographic distribution of these K. pneumoniae carbapenemase (KPC) enzymes in Enterobacteriaceae, mainly K. pneumoniae, was limited to the eastern part of the United States (6). Nowadays, the worldwide spread of KPC-producing, Gram-negative pathogens represents a potential clinical threat with devastating effects on patient outcomes (13).

Although KPCs are mostly identified in K. pneumoniae, they have recently been observed among other Gram-negative pathogens, such as Pseudomonas spp. in America (11, 14) and Acinetobacter baumannii in Puerto Rico (12). KPC-2-producing K. pneumoniae and Escherichia coli clinical isolates were also identified in Brazil (8, 9). Besides these, to date, only one report describes the isolation of Pseudomonas putida producing KPC-2 in Texas (2). Those few reports show the spreading of the KPC-2 enzyme among potential pathogenic bacteria, an observation which might be of great concern for infection control programs. In the present study, we report the identification of the first case of a KPC-2-producing P. putida isolate in Brazil and highlight the importance of this finding in terms of hospital infection control.

An 8-year-old boy was admitted to the Oncology Pediatric Center at the University Hospital Oswaldo Cruz, Recife, Brazil, in July 2008 to initiate a new cycle of chemotherapy for Burkitt's lymphoma. Ten days following admission, he presented fever and diarrhea and was sent to the Pediatric Intensive Care Unit and given a further 10 days of empirical intravenous antibiotic therapy with meropenem (1 g every 8 h) and vancomycin (500 mg every 6 h). The patient evolved with febrile neutropenia and gastrointestinal bleeding. Due to fungal sepsis by Candida spp., he received voriconazole (7 mg/kg of body weight every 12 h) for 21 days. Transcatheter bloodstream cultures showed the presence of a carbapenem-resistant P. putida isolate (herein named PP36). Since the patient has no history of traveling abroad, we assumed that this infection was acquired at the hospital. The bacterial identification was performed by a mini-Api ID32 GN card (bioMeriéux, Marcy l'Etoile, France). Meropenem therapy (1 g every 8 h) was restarted, and the catheter was removed. After the removal, the patient evolved to negative bloodstream cultures until the 67th day of internment when he was discharged.

Broth microdilution assays (Table 1) showed that the PP36 isolate was resistant to all antimicrobials tested according to Clinical and Laboratory Standards Institute protocols (4, 5), that it was susceptible to polymyxin B according to EUCAST breakpoints (7), and that it showed intermediate resistance to tigecycline according to U.S. Federal Drug Administration breakpoints for Enterobacteriaceae that define a MIC of ≤2 as susceptible. E. coli ATCC 25922 was used as the control. Due to the resistance to all beta-lactams tested, the isolate was screened for the presence of extended-spectrum beta-lactamases (ESBLs) and class A and B carbapenemases (Table 1). PCR amplifications and plasmid analysis were carried out as previously described (10), followed by sequencing of the amplicon in both strands (ABI 3100 platform; Applied Biosystems) and BLASTn analysis (www.ncbi.nlm.nih.gov/Blast.cgi). PCR-based replicon typing and transposon typing were performed as described (3, 6).

Table 1.

Antimicrobial drug susceptibility and molecular analysis of the bacterial strains used in this work

Straina	MIC (μg/ml) forb:														Presence of blaKPC-2/class 1 integronc	Plasmid size(s) (kb)
	GEN	CEF	CTX	CRO	CAZ	FEP	ATM	IPM	MEM	CIP	TZP	AMC	PMB	TGC
PP36	64	>256	256	128	16	256	128	64	>32	0.25	256	≥128	0.12	4	+/+	65, 147
TF36	64	>256	256	64	8	32	128	16	2	0.25	64	64	0.12	4	+/+	65
DH5α	0.25	8	<0.12	0.06	0.5	0.5	0.5	0.12	<0.015	0.004	<2	<1	<0.12	0.06	−/−
ATCC 25922	0.06	4	0.12	0.06	0.12	<0.12	0.12	0.12	0.015	0.008	<2	2	0.25	0.03	−/−

^aPP36, P. putida isolate; TF36, E. coli transformant of PP36; DH5α, E. coli recipient strain; ATCC 25922, E. coli control strain.

^bGEN, gentamicin; CEF, cephalothin; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; FEP, cefepime; ATM, aztreonam; IPM, imipenem; MEM, meropenem; CIP, ciprofloxacin; TZP, piperacillin-tazobactam; AMC, amoxicillin-clavulanic acid; PMB, polymyxin B; TGC, tigecycline.

^c+, present; −, absent.

Afterwards, genetic analysis resulted in the detection of the bla_KPC-2 gene. The detection of the intI1 gene by specific PCR showed the presence of a class 1 integron. Furthermore, its variable region of ca. 1.5 kb was submitted to DNA sequencing that revealed the presence of the dhfrXVb gene, encoding the dihydrofolate reductase involved in resistance to trimethoprim, and the new allele arr-6, encoding the rifampin ADP-ribosylating transferase involved in resistance to rifampin. This new allele differs from arr-2 by an A-to-G mutation at the position +293 (Lys98Arg) and from arr-3 by a silent G-to-A mutation at the position +411. The PP36 isolate displayed two distinct plasmids with sizes of ca. 65 kb and 147 kb (Table 1) that were extracted and introduced into Escherichia coli DH5α (10). The transformed cells, designated TF36, that were selected on Mueller-Hinton agar containing 100 μg/ml ampicillin showed the presence of only the 65-kb plasmid that carried the bla_KPC-2 gene as attested by specific PCR. Recently, Andrade et al. (1) described the presence of the plasmid types IncFII, IncN, and IncL/M carrying bla_KPC among Enterobacteriaceae species in Brazil. In the present work, we identified a 65-kb IncFI-type plasmid carrying bla_KPC. Acquisition of this plasmid yielded resistance to aztreonam, cephalosporins, carbapenems, and combinations of beta-lactam/beta-lactamase inhibitors to the transformed cells (Table 1). It was noteworthy that MICs for ceftriaxone and imipenem increased 64- and 134-fold, respectively, in the TF36 strain compared to those of the DH5α strain.

The analysis of the genetic environment of bla_KPC by amplification of the region between ISKpn7 and the bla_KPC gene revealed a 200-bp deletion upstream of the bla_KPC gene. It indicates the presence of a Tn4401c isoform located in the 65-kb IncFI plasmid. Since KPC-producing K. pneumoniae isolates are frequent in the hospital of study, it is possible that P. putida acquired a bla_KPC-containing plasmid from these isolates. This report confirmed for the first time in Brazil the presence of a plasmid-based bla_KPC-2 gene in a hospital isolate of P. putida. Moreover, a new allele for rifampin resistance was identified. Despite careful and conservative use of antibiotics and good control practices, the present work and the recent identifications of the bla_KPC genes in distinct Gram-negative pathogens emphasize the quick spreading of this gene in long-term facilities, limiting therapeutic options for infected hospitalized patients.

Nucleotide sequence accession numbers.

The sequences for the newly detected bla_KPC-2 gene, dhfrXVb gene, and arr-6 allele have been deposited in GenBank under accession numbers JF922884, JF922882, and JF922883, respectively.