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Antimicrob Agents Chemother. 2010 March; 54(3): 1354–1357.
Published online 2009 December 28. doi:  10.1128/AAC.00899-09
PMCID: PMC2825984

Detection of KPC in Acinetobacter spp. in Puerto Rico [down-pointing small open triangle]

Abstract

During an island-wide PCR-based surveillance study of beta-lactam resistance in multidrug-resistant (MDR) Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus-baumannii complex isolates obtained from 17 different hospitals, 10 KPC-positive Acinetobacter isolates were identified. DNA sequencing of the blaKPC gene identified KPC-2, -3, and -4 and a novel variant, KPC-10. This is the first report of a KPC-type beta-lactamase identified in Acinetobacter species.

The Acinetobacter calcoaceticus-baumannii complex has been recognized, during the last few decades, as an important opportunistic pathogen associated with life-threatening nosocomial infections and hospital outbreaks, as well as an agent of serious infections in injured U.S. military personnel returning from the Middle East war zones (9, 25, 33). In addition to their ability to survive in adverse environmental conditions (14), the organisms have intrinsic and acquired mechanisms of antimicrobial resistance, such as porin downregulation, overexpression of efflux pumps, chromosomal and plasmid-acquired beta-lactamases, aminoglycoside-modifying enzymes, and fluoroquinolones resistance, among others (11). Class B metallo-beta-lactamases and class D oxacillinase with carbapenemase activity have been identified in Acinetobacter species as a mechanism of broad-spectrum beta-lactam resistance (11, 20).

The molecular class A beta-lactamases of the KPC family are a group of potent carbapenemases identified initially in a Klebsiella pneumoniae isolate from the United States and later in other members of the Enterobacteriaceae family and in other geographical regions worldwide (17, 20). Pseudomonas aeruginosa positive for the blaKPC gene has been recently identified in Colombia, Puerto Rico, and Trinidad and Tobago (3, 28, 29).

Up to date, eight different KPC variants (KPC-2 to -9) have being identified differing by 1 or 2 two amino acid substitutions. KPC-2 and -3 are the most common variants identified in Enterobacteriaceae and P. aeruginosa. KPC-6, -7, and -8 have been identified only in K. pneumoniae, while KPC-9 was detected in Escherichia coli and KPC-5 in P. aeruginosa. All the KPC variants except for KPC-7 and -9 have been detected in Puerto Rico (21-24, 29). In this report, we describe for the first time the presence of the KPC gene in clinical isolates of Acinetobacter species in Puerto Rico and the identification of a novel KPC variant, KPC-10, in one of the isolates.

A PCR-based surveillance study of beta-lactam resistance was started in January 2009 in 17 hospitals across the island. After Institutional Review Board approval, the participating hospitals sent isolates of all unique, consecutive, multidrug-resistant Acinetobacter species with their corresponding susceptibility report and basic epidemiologic information. PCR screening with family-specific beta-lactamase primers for KPC and IMP and multiplex PCR for OXA carbapenemases and for CTX-Ms were performed as previously described (29, 31, 32). The Vitek DCS-R5 system confirmed the identification of the isolates as those in the A. calcoaceticus-baumannii complex. Bidirectional sequencing of the full-length blaKPC gene PCR product was independently generated at least twice to identify the type of the KPC variant. DNA sequencing was commercially performed by Davis Sequencing. Sequence alignment and analysis were done online by utilizing the BLAST program (www.ncbi.nlm.nih.gov). Trek GNXF Gram-negative MIC plate microdilution panels (Westlake, OH) were utilized to perform antibiotic susceptibility tests by following the manufacturer's instruction, and the results were interpreted as recommended by the Clinical and Laboratory Standards Institute (8). No attempts were made to evaluate patients' therapies or clinical outcomes.

From a total of 274 multidrug-resistant Acinetobacter isolates collected from January to May of 2009, 10 (3.4%) were identified as KPC positive. Seven were detected in the metropolitan San Juan area, two in the north, and one in the central region of Puerto Rico.

Table Table11 shows the patients' pertinent clinical information and type of KPC variants identified. There were six male and four female patients, whose average age was 69 years, ranging from 40 to 95 years. Six were isolated from patients in the intensive care units either from sputum (four isolates) or blood (two isolates) samples. The average hospital length of stay was 41.6 days (range, 11 to 96 days). Six patients had ventilator-associated pneumonia with or without sepsis. Six patients had skin and soft-tissue infections. All patients had significant comorbid conditions, such as cardiovascular, renal, neurologic, or traumatic injuries. The crude mortality rate for the 10 patients was 40%. Nine patients were treated with multiple antibiotic courses during their hospitalization (data not shown). Our patients shared similar characteristics to those described in the literature, such as significant comorbidities, prolonged hospitalizations, invasive procedures, exposure to multiple antibiotics regimes, and high crude mortality rates (1, 2, 10, 27). In our island-wide surveillance study, all hospitals with KPC-positive Acinetobacter isolates also had KPC-positive Klebsiella pneumoniae, Pseudomonas aeruginosa, and/or Escherichia coli isolates (21).

TABLE 1.
Pertinent clinical information and type of KPC variants

The susceptibility tests shown in Table Table22 showed that colistin, polymyxin B, tigecycline, and minocycline had the lowest MICs of the tested antibiotics. Resistance to quinolones, aminoglycosides, and to the beta-lactam antibiotics, including carbapenems, was observed. The therapeutic options available for the treatment of multidrug-resistant Acinetobacter are limited (5, 15). Our results, as well as others (4, 7, 13, 26, 34), suggested that tigecycline, polymyxins, colistin, and possibly minocycline have consistent in vitro activity against this organism.

TABLE 2.
MICs of 10 KPC-positive Acinetobacter calcoaceticus-baumannii complex isolates to selected antibiotics

The PCR results obtained with family-specific beta-lactamase primers showed that all isolates were positive for blaKPC and blaOXA-51-like groups and negative for blaIMP and blaCTX-M genes. Sequencing of blaKPC detected the following variants: KPC-3 in seven isolates; KPC-4, KPC-2, and a novel KPC-10 (GenBank accession number GQ140348) in one isolate each. The identification of a class D blaOXA-51-like gene in all isolates was expected, since it had been shown to be chromosomally located in A. baumannii (11, 20). One isolate had the blaOXA-58-like group, which has been shown to be carried on a plasmid, recovered from diverse geographic regions, and associated with nosocomial outbreaks (6, 12, 18, 19).

To our knowledge, this is the first report of multidrug-resistant A. calcoaceticus-baumannii complex clinical isolates harboring the KPC gene. The presence of this gene suggests the possibility of horizontal transmission, as this carbapenemase has been associated with mobile genetic elements (transposons) which can be transferred from one bacterium to another (11, 16, 17, 30).

The presence of the blaKPC gene in Acinetobacter species adds another important element to an organism already harboring multiple innate and acquired mechanisms of resistance with the real possibility of horizontal transfer of a very troublesome and potent carbapenemase. This clearly emphasizes the importance of judicious use of antibiotics and strict infection and environmental control practices in acute and chronic care facilities to reduce the possibility of nosocomial transmission and potential outbreaks.

Acknowledgments

This work was supported by Janssen Ortho-McNeil, Inc. (Johnson & Johnson); Merck Sharp and Dohme, Inc.; Pfizer, Inc.; and NCRR/NIH-RCMI award G12RR03051.

We appreciate the technical assistance of Caleb Fernandez, Teresa Martínez, Iván Claudio, Yanira Rosario, Melissa Miranda, Tania Diaz, and Jennifer Pérez and also Wieslaw J. Kozek for reviewing the manuscript. We thank the following hospital medical technologists for collecting the samples and clinical information: Myriam Corazón, María Matos, Linnette Santos, Nayda Vázquez, and Abigail Torres.

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 December 2009.

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