Emergence of Klebsiella pneumoniae clinical isolates producing KPC-2 carbapenemase in Cuba

The emergence of Klebsiella pneumoniae producing carbapenemase (KPC) has now become a global concern. As a part of a nationwide multicentre surveillance study in Cuba, three K. pneumoniae clinical isolates resistant to carbapenems were detected for a 1-month period (September to October 2011). PCR and sequence analysis revealed that the three strains harboured blaKPC-2. They showed resistance or intermediate susceptibility to expanded-spectrum cephalosporins, other β-lactams, a β-lactam/β-lactamase inhibitor combination, and gentamicin. Two strains were susceptible only to colistin, whereas the other strain showing colistin resistance was susceptible to fluoroquinolones. These blaKPC-2-positive K. pneumoniae strains were classified into ST1271 (CC29), a novel clone harbouring blaKPC-2, and were revealed to be genetically identical by PCR-based DNA fingerprinting. The three patients infected with the KPC-producing K. pneumoniae had common risk factors, and had no overseas travel experience outside Cuba, suggesting local acquisition of the resistant pathogen. This is the first report of a KPC-producing K. pneumoniae in Cuba. Although detection of KPC in Enterobacteriaceae is still rare in Cuba, our finding indicated that KPC-producing bacteria are a global concern and highlighted the need to identify these microorganisms in clinical laboratories.


Introduction
The Klebsiella pneumoniae carbapenemase (KPC) was first described in 1996 in North Carolina, USA. Thereafter, expansion of KPC in clinical isolates has been reported from different continents associated with the global spread of the clonal lineages of K. pneumoniae, such as ST248/ST258 [1]. The ST258 K. pneumoniae has been described as an international KPC-producing clone [2], and its global spread including Latin American countries has also been reported [3]. Because the KPC gene is carried by plasmids, potentially rapid transmission of carbapenem resistance has been recognized as a major threat to the antimicrobial treatment of infections with gram-negative microorganisms [2]. The Pan American Health Organization issued an epidemiological alert with the increase of carbapenemase in Enterobacteriaceae from many Latin American countries in 2010 [4]. Since then, Cuba has initiated a surveillance network for K. pneumoniae clinical isolates from reference hospitals to analyse their antimicrobial susceptibility and genetic mechanisms of drug resistance with special attention to carbapenem resistance, in the 'Pedro Kour ı' Institute of Tropical Medicine [5]. During this national surveillance, three isolates of KPC-producing K. pneumoniae were detected for the first time in Cuba.

Methods
As a part of a nationwide multicentre surveillance study in Cuba, three K. pneumoniae clinical isolates resistant to carbapenems were detected in the 'Pedro Kour ı' Institute of Tropical Medicine for a 1-month period (September to October 2011). The antimicrobial susceptibility to a wide range of antibiotics was determined using E-test (BioM erieux, Marcy l'Etoile, France) according to the manufacturer's recommendation. MICs were interpreted into susceptible or resistant according to CLSI guidelines, 2012 [6], except for colistin, which was judged by EUCAST criteria (susceptible ≤2 g/mL, resistant ≥4 g/mL), (http://www.eucast.org/clinical breakpoints/). A double-disc synergy test was performed to detect extended spectrum b-lactamases (ESBLs) [6] and 3aminophenylboronic acid test was used to screen for production of carbapenemases [7]. The presence of genes encoding carbapenemase was determined by PCR using protocols and conditions as described previously [8]. Nucleotide sequence of bla KPC was determined by direct sequencing with PCR products by using the BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequence type (ST) of K. pneumoniae based on the scheme of multilocus sequence typing (MLST) was determined according to the methods available at the website (www.pasteur.fr/ recherche/genopole/PF8/mlst/Kpneumoniae.html).

Results and Discussion
Three clinical isolates of K. pneumoniae (strains 328, 354, 355) recovered from two provinces of Cuba (Holguin and Havana city) were confirmed to be resistant to imipenem and meropenem. These strains showed resistance or intermediate susceptibility to expanded-spectrum cephalosporins, b-lactam, b-lactam/b-lactamase inhibitor combination (piperacillin/tazobactam), and gentamicin. Two of these strains (354, 355) were susceptible only to colistin, whereas the other strain (328) showing resistance to colistin was susceptible to fluoroquinolones ( Table 1). The synergy test to detect ESBLs and carbapenemase was positive, suggesting the production of an ESBL and KPC enzyme. The KPC gene was detected in the three strains by PCR, and their sequences were revealed to be identical to bla KPC-2 . The three bla KPC-2 -positive K. pneumoniae strains were classified into ST1271 (CC29), a single locus variant of ST29. The ST1271 was identified as a novel clone harbouring bla KPC-2 in the present study, because the presence of bla KPC-2 had not been reported in ST1271 as well as ST29 clones. By randomly amplified polymorphic DNA analysis with five different primers and REP PCR, three strains showed the same banding patterns (Fig. 1), indicating that these strains are genetically identical and have the same origin. The two colistin-susceptible strains were derived from the same hospital in Havana, exhibited similar resistance patterns, and are suggested to be the same strain that had transmitted via nosocomial infection.
The three strains with KPC genes were derived from two separate provinces, i.e. Holguin (eastern Cuba) and Havana city (western Cuba). Patients infected with the KPC-producing K. pneumoniae had no experience of travel outside Cuba, suggesting that local acquisition and a silent dissemination of the KPC-positive K. pneumoniae ST1271 clone in this country. Because intercontinental travel has been directly linked with the spread of KPCs through patients colonized or infected with KPC-producing K. pneumoniae [14,15], international tourism might have played an important role in its emergence in Cuba. ST29 K. pneumoniae strains have been isolated from Europe, e.g. Spain and Greece, and also from Brazil [3]. In our previous report on the antimicrobial resistance of K. pneumoniae in Cuba through the national surveillance programme (2009-10), 54 isolates (23.6%) were positive for ESBL, and were classified into 27 STs, showing high clonal diversity [5]. However, none of the ST identified in the ESBL-positive isolates belonged to CC29. Therefore, in Cuba, the ST1271 K. pneumoniae strains are genetically distinct from ESBL-positive strains. Although the origin of the ST1271 strains has yet to be determined, it is possible that ST1271 might have been brought from another country recently, associated with acquisition of the KPC gene. Further molecular epidemiolog- ical studies on K. pneumoniae isolates, including those from healthy individuals, may provide more information on distribution and transmission of the ST1271 clone. It was noted in the present study that an ST1271 K. pneumoniae strain showed resistance to colistin. Generally, the colistin resistance rate of K. pneumoniae has been reported to be low [16]. During the period of antimicrobial resistance surveillance (2010-12) of Klebsiella spp. in Cuba, we notified 15% colistin resistance, which would be of great concern in clinical settings [17]. Our present finding indicates that the colistin resistance has emerged in multidrug-resistant K. pneumoniae. This antibiotic is recognized as a key therapeutic option for carbapenem-resistant bacteria, and is particularly important in countries with limited resources, such as Cuba where tigecycline is not available. Table 2 shows the clinical information of three patients infected with K. pneumoniae producing KPC-2 carbapenemase. These patients had common risk factors, such as prolonged hospitalization, intravenous catheter, previous antimicrobial therapy, and underlying disease. These findings are consistent with previous studies that notified risk factors for acquiring infections with KPC-producing K. pneumoniae [18]. In the present study, all three K. pneumoniae producing KPC were isolated from blood specimens and two of the patients died  with septic shock. The susceptibility profile of these isolates indicated considerably limited therapeutic options, i.e. colistin in two patients or fluoroquinolone in one patient. The optimal treatment for infections caused by KPC-producing K. pneumoniae has been difficult to describe, resulting in mortality rates of at least 50% [19]. Therefore, evaluation of effective antibiotic options and rigorous infection control measures may be necessary to reduce carbapenemase-producing microorganisms.
Detection of the novel KPC-producing K. pneumoniae clone (ST1271) in the present study demonstrated the importance of monitoring hospitalized patients for the further emergence of carbapenem resistance in K. pneumoniae as well as in other gram-negative pathogens. Although carbapenemase-producing Enterobacteriaceae are still rare in Cuba, our finding confirmed that KPC-producing isolates are a global concern, highlighting the need to identify these microorganisms in clinical laboratories.