IMP-16 in Pseudomonas putida and Pseudomonas stutzeri: potential reservoirs of multidrug resistance

Production of metallo-β-lactamases (MBLs) is one of the leading carbapenem resistance mechanisms among Gram-negative aerobes. The IMP- and VIM-type enzymes are considered the most widespread as they have been reported from several continents. These enzymes are very often encoded by integron-borne genes with several allelic variants known for each type (Queenan & Bush, 2007). While SPM is the most common MBL type in Brazil and a Pseudomonas aeruginosa epidemic clone has been detected in several Brazilian cities (Gales et al., 2003; Carvalho et al., 2006), IMP and VIM variants have also been described in some isolates of P. aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae (Sader et al., 2005; Penteado et al., 2009). In this study, we report the presence of MBL IMP-16 in carbapenem-resistant Pseudomonas putida and Pseudomonas stutzeri isolates recovered from two children who attended two different hospitals in Rio de Janeiro, Brazil.

P. putida and P. stutzeri are environmental species reported as a rare cause of clinical infection. Infections caused by these agents have been described in immunosuppressed hosts or patients with invasive medical devices (Yee-Guardino et al., 2006; Aumeran et al., 2007). They should be considered pathogenic when isolated from pure culture. Unlike P. aeruginosa, they are usually susceptible to a variety of antibiotics (Sader & Jones, 2005).

P. stutzeri CCBH 4919 was isolated in March 2009 from cerebrospinal fluid (CSF) of a 7-year-old female patient in the intensive care unit (ICU) of the Prontobaby Hospital da Criança, a private paediatric hospital. The patient had a brain tumour and was admitted for surgery and insertion of external ventricular derivation in February 2009. Complications arose when she developed meningitis 23 days post-surgery. The patient initially received intravenous (i.v.) meropenem and amikacin. Intrathecal amikacin doses (5 mg per day) were initiated 9 days after i.v. antibiotic therapy was prescribed due to persistent clinical symptoms and recurrent isolations of P. stutzeri in the CSF. Intrathecal doses were increased to 7.5 mg per day and the child received a total of 27 days of intrathecal and i.v. amikacin, 20 days of meropenem and 14 days of ciprofloxacin, with three consecutive negative CSF cultures. No side effects were attributed to intrathecal therapy. The child was discharged about 2.5 months after admission on 18 April 2009 with no neurological sequelae.

Few cases involving P. stutzeri in the central nervous system have been described in the English literature. One case occurred in a man infected with HIV who underwent antimycobacterial therapy for tuberculous lymphadenitis and whose CSF grew P. stutzeri after he presented with symptoms of meningitis (Roig et al., 1996). Another case was in a child who had a hospital-acquired P. stutzeri brain abscess after subdural grid implantation before surgery for refractory epilepsy (Yee-Guardino et al., 2006).

P. putida CCBH 4308 was recovered in June 2008 from blood of a 2-month-old female patient with interatrial communication hospitalized due to bronchiolitis in the paediatric ICU of Hospital dos Servidores do Estado, a tertiary public hospital. The patient responded clinically to ampicillin and gentamicin (i.v.) for 10 days and was discharged 12 days after P. putida isolation.

Species identification was realized by the API 20NE system (bioMérieux) and confirmed by 16S rRNA gene sequencing. The MICs of ceftazidime, cefotaxime, imipenem, meropenem, gentamicin, amikacin and polymyxin B were obtained using Etest (AB Biodisk). P. putida CCBH 4308 and P. stutzeri CCBH 4919 were resistant to all tested β-lactams and susceptible only to amikacin, gentamicin and polymyxin B (Table 1⇓). The β-lactam resistance pattern was unusual for members of these species (Sader & Jones, 2005). In particular, resistance to carbapenems suggests the possibility of carbapenemase production or another acquired resistance mechanism. In fact, MBL production was detected in both isolates using a ceftazidime–2-mercaptopropionic acid double-disc synergy test and imipenem–EDTA combined-disc test. The isolates showed PCR-positive results by using bla_IMP-like primers; and amplicon sequencing revealed 100 % sequence identity with bla_IMP-16. P. putida and P. stutzeri isolates were positive for the class 1 integron (Lévesque et al., 1995) with amplicon lengths of about 2.3 kb and 1 kb, respectively, corresponding to the size of the variable region. DNA sequencing of the variable region revealed that the P. stutzeri integron carried bla_IMP-16 only, and the P. putida integron carried three gene cassettes (aacA7, aacA4 and aadA1) that confer resistance to aminoglycosides. However, the bla_IMP-16 gene of P. putida was not located in this mobile element. The possibility exists that this gene is located on another undetected mobile element.

The presence of plasmid DNA was not observed in the P. putida isolate using a Midi preparation of plasmid DNA by alkaline lysis with SDS, as described by Sambrook & Russell (2001). The P. stutzeri isolate harboured a unique plasmid estimated to be about 2.3 kb in size. Southern blot hybridizations with digoxigenin-labelled bla_IMP-16 generated by use of the PCR DIG detection system (Roche Diagnostics) were performed to determine the location of the MBL gene. The bla_IMP-16 probe did not hybridize to plasmid extracts from P. stutzeri, suggesting that this gene was carried on the chromosome in both isolates.

An IMP-16 variant has been described before in a P. aeruginosa strain isolated from a 60-year-old man in a hospital in Brasília city, Brazil (Mendes et al., 2004). The bla_IMP-16 gene was found in a class 1 integron with a 2.9 kb variable region located on the chromosome of P. aeruginosa. To our knowledge, bla_IMP-16 has only been described in the literature in this index strain. Our report is the first description of IMP-16 production in P. stutzeri and P. putida isolates.

Little is known about chromosomal and transferable carbapenem resistance in environmental Pseudomonas species, although MBLs have been reported particularly in P. putida (Juan et al., 2010). In P. stutzeri, this is the second description of an IMP-type MBL. The first description was in Taiwan (Lee et al., 2004). Our findings suggest that the environmental organisms acquired the MBL genes probably under selective pressure of antibiotic exposure in the hospital environment, and highlight the threat as a potential reservoir of MBLs and other resistance genes, corroborating what has been described previously (Juan et al., 2010). Hence, hospital surveillance for MBL-producing isolates in environmental reservoirs is warranted.