Detection of an international multiresistant clone belonging to sequence type 654 involved in the dissemination of KPC-producing Pseudomonas aeruginosa in Argentina

Sir, The emergence of Klebsiella pneumoniae carbapenemase (KPC) has now become a global concern. KPC producers are mostly Enterobacteriaceae, but Pseudomonas aeruginosa have also been reported and mostly identified in the American continent.1–4 However, it is unknown if this is due to the spread of epidemic strains, since the multilocus sequence type (ST) has not been provided in most of those reports. The aim of this work was to characterize KPC-producing P. aeruginosa isolated in Argentina from 2006 to June 2011. Since 2005, we designed an algorithm to detect carbapenemases (metallo-β-lactamases, KPC, etc.) in P. aeruginosa at the level of the clinical microbiology laboratory, which was implemented among 432 hospitals. All P. aeruginosa were screened through that algorithm, and KPC production was suspected in isolates with high-level resistance to carbapenems and aztreonam (absence of disc zones) and a negative synergy test result between the carbapenems and EDTA, a phenotype consistent with the reported patterns of KPC-producing P. aeruginosa.1,4 As a result, 65 isolates were suspected to be KPC producers (Table 1). Strains were isolated from nine hospitals (seven cities, five provinces), six of which were located in the Patagonia region. The first KPC producer was detected in a hospital from Bariloche in 2006. Dissemination to other locations (except Chaco) was associated with patients previously hospitalized in Bariloche (Table 1). Epidemiological and molecular characteristics of KPC-positive P. aeruginosa isolates INEI, Instituto Nacional de Enfermedades Infecciosas. aWHONET AR, WHONET Argentina Network (90 Hospitals, 24 Provinces); SSC, Sanatorio San Carlos; HPR, Hospital Privado Regional del Sur y Sanatorio del Sol; BAR, Hospital Zonal de Bariloche; HZLP, Hospital Zonal de Lago Puelo; CGR, Laboratorio Roca, Clínica Roca; HZE, Hospital Zonal de Esquel; HPC, Hospital Privado Centro Modelo de Córdoba; UDA, Hospital Municipal de Gastroenterologia Udaondo; HJP, Hospital Julio Perrando. bIsolates suspected of producing KPC defined with the phenotypic algorithm indicated in the text. The denominator represents the total P. aeruginosa isolates recovered in the indicated hospital(s) of the corresponding row. cAs defined by agar dilution according to CLSI. S, susceptible; CST, colistin; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin. The number of isolates is shown in parentheses. dBaseline obtained from the WHONET Argentina Network. eNA, not applicable. fThe respective index patients of these locations were previously hospitalized in Bariloche. gJanuary–June 2011. Epidemiological and molecular characteristics of KPC-positive P. aeruginosa isolates INEI, Instituto Nacional de Enfermedades Infecciosas. aWHONET AR, WHONET Argentina Network (90 Hospitals, 24 Provinces); SSC, Sanatorio San Carlos; HPR, Hospital Privado Regional del Sur y Sanatorio del Sol; BAR, Hospital Zonal de Bariloche; HZLP, Hospital Zonal de Lago Puelo; CGR, Laboratorio Roca, Clínica Roca; HZE, Hospital Zonal de Esquel; HPC, Hospital Privado Centro Modelo de Córdoba; UDA, Hospital Municipal de Gastroenterologia Udaondo; HJP, Hospital Julio Perrando. bIsolates suspected of producing KPC defined with the phenotypic algorithm indicated in the text. The denominator represents the total P. aeruginosa isolates recovered in the indicated hospital(s) of the corresponding row. cAs defined by agar dilution according to CLSI. S, susceptible; CST, colistin; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin. The number of isolates is shown in parentheses. dBaseline obtained from the WHONET Argentina Network. eNA, not applicable. fThe respective index patients of these locations were previously hospitalized in Bariloche. gJanuary–June 2011. A subset (n = 30) of suspected isolates was submitted to the National Reference Laboratory (Malbrán Institute), where blaKPC-2 was confirmed in all strains by PCR/sequencing. XbaI PFGE analysis, using previously described criteria,5 revealed that all but one of the isolates belonged to a single pulsotype (Table 1). In Argentina, these PFGE patterns had not previously been observed. Multilocus sequence typing of one strain of each PFGE type revealed that the dominant clone (PFGE type A) belonged to ST654, while the unique strain of PFGE type B (Chaco), belonged to ST162 (http://pubmlst.org/paeruginosa) (Table 1). To investigate the genetic organization of blaKPC-2 in P. aeruginosa, we used PCR primer pairs located in the Tn4401 structure and in the flanking sequences found in Enterobacteriaceae with KPC in our country, called Variant 1a (ISKpn8 and ISKpn6-like).6 All isolates belonging to ST654 harboured blaKPC-2 in Tn4401b, which was confirmed by sequencing. The ST162 isolate harboured blaKPC-2 in Variant 1a.6 By plasmid content analysis and Southern blotting with a blaKPC-2 probe,6 we found that ST654 contains blaKPC in a plasmid of 50 kb, while the strain ST162 contains several plasmids but only one of 47 kb hybridized with KPC. Plasmid incompatibility (Inc) groups were further analysed using the PCR-based replicon-typing protocol described for Enterobacteriaceae.7 Both ST162 and ST654 gave negative results for all the Inc groups tested. The strains were highly resistant (MICs >128 mg/L) to aztreonam, cefepime, imipenem, meropenem and piperacillin/tazobactam by agar dilution. Colistin (MICs 1 mg/L), gentamicin (MIC90s 2 mg/L) and amikacin (MIC90s 4 mg/L) were the most active drugs. ST162 was susceptible to ciprofloxacin (MIC 0.5 mg/L) (Table 1). The origin of blaKPC-2-possessing ST654 remains uncertain. The mobilization of blaKPC from Enterobacteriaceae was ruled out: first, these strains were not detected in Bariloche until 2010 and, second, they harboured blaKPC-2 in the Variant 1a.6 ST162 was isolated from a patient with no recent history of travel. Interestingly, this patient shared the same ward simultaneously with two other patients undergoing infections due to KPC-producing Enterobacter cloacae and K. pneumoniae. In these isolates, the blaKPC-2 genetic environment matched that of ST162. However, blaKPC-2 in E. cloacae and K. pneumoniae was associated with plasmids of different sizes (40 and >150 kb, respectively) and different Inc groups (N and FIIS, respectively)6 from that detected in ST162. Thus, we speculated that the surge of ST162 could be due to blaKPC-2 mobilization among different plasmids (i.e. transposition). Clonal complexes CC111 and CC235 have been reported as the major clones involved in the global dissemination of extended-spectrum and metallo-β-lactamases in P. aeruginosa.8 However, we observed that the main dissemination of KPC was mediated by a new clone (ST654) not related to both CC111 and CC235. ST654 is endemic in Singapore,9 where it has been associated with IMP-1 and IMP-26; it has also been reported in Sweden as a VIM-2 producer, although the isolate was imported from Tunisia.10 Unlike other Latin American regions, such as Puerto Rico and Colombia, where different PFGE types and STs have been identified,1,4 the dissemination of KPC-producing P. aeruginosa in Argentina is mainly associated with a single clone. These findings confirm that ST654 plays an important role in the global spread of carbapenemases, either metallo-β-lactamases or KPC. Thus, the worldwide dissemination of KPC-producing P. aeruginosa of ST654 might be expected and should be monitored. Sanatorio San Carlos, Bariloche, Río Negro, Argentina: Barbara Bavdaz; Hospital Privado Regional del Sur y Sanatorio del Sol, Bariloche, Río Negro, Argentina: Gabriela Rubinstein; Hospital Zonal de Bariloche, Bariloche, Río Negro, Argentina: Nestor Blazquez; Hospital Zonal de Lago Puelo: Pablo Nouveau; Laboratorio Roca, Clínica Roca, General Roca, Rio Negro, Argentina: Silvana Cecchi; Hospital Zonal de Esquel, Chubut, Argentina: Omar Daher; Hospital Privado Centro Modelo de Córdoba, Córdoba: Mario Vilaró; Hospital Municipal de Gastroenterología ‘B. Udaondo’, Ciudad Autónoma de Buenos Aires, Argentina: Claudia Brotto; and Hospital Julio Perrando, Resistencia, Chaco, Argentina: Isabel Marques. This work was supported by the regular federal budget of the Ministry of Health of Argentina. None to declare. Part of this work was presented at the Fifty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2011 (Abstract no. C2-1700).