Identification of KPC-2-producing Pseudomonas aeruginosa isolates in China

Sir, KPCs have been reported in different parts of the world since the first identification of KPC-positive Klebsiella pneumoniae in the USA.1,2 The increasing emergence and spread of KPCs leaves fewer available therapeutic options due to their broad-spectrum hydrolytic activity and high mobility.2 The KPC-producing bacteria are mostly Enterobacteriaceae, but also rarely Pseudomonas aeruginosa and Acinetobacter spp.2 The first KPC-producing P. aeruginosa isolates were identified in Colombia.3 Although there have been several reports of KPC-2-producing Enterobacteriaceae in China in recent years, this is the first report of KPC-producing P. aeruginosa in China. In a surveillance study for assessing asymptomatic colonization with carbapenem-resistant organisms in the intensive care unit (ICU) of a 2200-bed teaching hospital (our hospital, i.e. First Affiliated Hospital, College of Medicine, Zhejiang University) in Hangzhou, 291 faecal specimens or rectal swabs were collected from 139 patients on admission and at 5day intervals after hospitalization and cultured using MacConkey agar plates (bioMérieux, France) containing 1 mg/L meropenem. Species identification was performed using the VITEK 2 system (bioMérieux, France). Ambler class A and B β-lactamase genes were detected for carbapenem-resistant organisms by PCR. Three KPC-2-producing P. aeruginosa isolates (P. aeruginosa 1–3) were recovered from three patients (patients 1–3) who occupied adjacent beds. Patient 1 was transferred from a regional hospital to our hospital's ICU with severe pneumonia in August 2009 and was discharged in September 2009. Patients 2 and 3 were admitted to our hospital's ICU with severe acute pancreatitis in August and September 2009, respectively. Patient 2 was discharged, but patient 3 died in September 2009. Mechanical ventilation was required for each patient. Neither patient 2 nor 3 had a recent history of hospitalization. None of the patients had a history of travel abroad. Each patient had extensive antibiotic exposure, including levofloxacin, cefepime, cefoperazone/sulbactam, piperacillin/tazobactam, meropenem and vancomycin. P. aeruginosa 1 and a carbapenem-resistant Acinetobacter baumannii (CRAB) were recovered from patient 1's rectal swabs 36 h after his admission to our hospital. For patient 2, a CRAB and a carbapenem-resistant Escherichia coli (CREC) were isolated from rectal swabs 21 days after admission and P. aeruginosa 2 was recovered from stools 25 days after admission. From the stools of patient 3, P. aeruginosa 3, a CRAB and a CREC were selectively isolated 7 days after admission. CRAB and CREC recovered from these three patients were KPC-negative. Susceptibility testing by Etest (bioMérieux, France) showed that all three P. aeruginosa isolates were pan resistant (resistant to all β-lactams, all aminoglycosides, all tetracyclines, all fluoroquinolones and polymyxin) and shared the same antimicrobial susceptibility patterns for the carbapenems (imipenem, meropenem and ertapenem; MICs >32 mg/L), polymyxin B (MIC = 16 mg/L) and colistin (MIC = 8 mg/L). blaPER, aac(3′)-II, aac(6′)-II and rmtB genes were co-identified from each P. aeruginosa isolate by using PCR assays followed by sequencing of Ambler class A, B, C and D β-lactamase genes, 16S rRNA methylase genes and aminoglycoside acetyltransferase genes. No metallo-β-lactamase (MBL) genes were detected. MBL (VIM-2, IPM-9 and IPM-1)-producing P. aeruginosa isolates are disseminated in different areas of China; however, the prevalence of MBL-positive P. aeruginosa isolates is lower than that of some developed countries.4 PFGE patterns of SpeI-digested genomic DNA showed the close relatedness of these three P. aeruginosa isolates.5 Multilocus sequence typing also revealed that these three isolates shared the same sequence type of ST463 (http://pubmlst.org/paeruginosa), which had not been identified in our hospital previously (Z. Ruan and T. Qu, Zhejiang University, personal communication). Conjugation experiments performed in mixed-broth cultures with the recipient strain of rifampin-resistant EC600 or sodium azide-resistant J53 failed. An ∼50 kb plasmid was extracted from each P. aeruginosa isolate using a Qiagen Plasmid Midi Kit (Qiagen, Germany). Repeated transformation experiments with the recipient strain of E. coli TOP 10 or E. coli DH5α also failed. Southern blotting experiments using the [α-33P]dCTP (DuPont, USA) labelling probes were performed to clarify the localization of blaKPC-2.6 For each P. aeruginosa isolate, the blaKPC-2-specific probe was unable to hybridize with the plasmid (Figure 1a and b), while it co-hybridized with one of four I-CeuI (New England Biolabs Inc., UK)-digested chromosomal bands (separated by PFGE), which hybridized with the P. aeruginosa 23S rRNA gene-specific probe (Figure 1c–e), indicating the chromosomal localization for this blaKPC-2 gene. Localization of the blaKPC-2 gene in three P. aeruginosa isolates. Electrophoretic profile of plasmids derived from three KPC-2-producing P. aeruginosa isolates (a) and Southern blotting using a blaKPC-2-specific probe (b). PFGE pattern of chromosome fragments after I-Ceu I digestion of three KPC-2-producing P. aeruginosa isolates (c) and Southern blotting using a P. aeruginosa 23S rRNA gene-specific probe (d) and a blaKPC-2-specific probe (e). Lanes: 1–3, P. aeruginosa isolates 1–3; 4, blaKPC-2-harbouring plasmids derived from a K. pneumoniae isolate preserved in the State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, College of Medicine, Zhejiang University (positive control); 5–7, P. aeruginosa isolates 1–3. A 2646 bp nucleotide segment encoding three open reading frames (ORFs) with the order of ISKpn8, blaKPC-2 and ISKpn6-like was identified by PCR mapping, as previously described.6 Sequences upstream and downstream of the 2646 bp fragment remain unclear. This 2646 bp structure was 100% identical to variant 2 of the blaKPC-2-flanking structure in plasmid pKP048, recently identified in Enterobacteriaceae isolates from several hospitals in China, indicating probable transmission of blaKPC-2 from Enterobacteriaceae to P. aeruginosa and its transfer between plasmid and chromosomal DNA.6 After the recent identification of KPC-producing P. aeruginosa isolates in South America, the Caribbean islands and North America, the identification of these organisms in China indicates a potential worldwide spread of KPC-producing P. aeruginosa. Considering P. aeruginosa 1 was recovered from the admission screening, we speculate that patient 1 had probably been colonized before admission. Patients 1 and 2 were KPC-producing P. aeruginosa carriers when they were discharged from our hospital, requiring active surveillance to detect and prevent the dissemination of this clone in China, especially in Zhejiang. This work was supported by research grants from the Ministry of Health of the People's Republic of China (no. 200802107) and Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents (no. 2007191). None to declare.