Molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolates from a university hospital in Brazil.

INTRODUCTION The emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kpn) isolates is attracting significant attention in nosocomial infection settings. K. pneumoniae is the main pathogen that harbours blaKPC genes. METHODOLOGY This study evaluated 54 K. pneumoniae carbapenem-resistant isolates from patients hospitalized at the University Hospital of Londrina, between July 2009 and July 2010. The isolates were phenotypically screened for carbapenemase production and submitted for genotypic confirmation by polymerase chain reaction (PCR) for KPC, metallo-β-lactamases, OXA-48, and extended-spectrum beta-lactamase genes. The absence of outer membrane proteins (OMP) was investigated by SDS-PAGE. The susceptibility profile was determined by broth microdilution, according to Clinical and Laboratory Standards Institute protocol. RESULTS All isolates were phenotypically positive for class A carbapenemase production, but negative for metallo-β-lactamase activity. PCR analysis demonstrated that all isolates carried blaKPC genes and sequencing showed that all strains belonged to KPC-2 subtype. Four strains did not show porin expression, and all isolates were resistant to ertapenem, meropenem, and imipenem. Susceptibility rates reached 35.2% for gentamicin, 85.2% for polymixyn B, 87% for colistin, and 98.1% for both tigecycline and fosfomycin. Pulsed-field gel electrophoresis showed six clones, and three of them predominated among the isolates. CONCLUSIONS KPC-2-producing K. pneumoniae is becoming predominant among carbapenem-resistant K. pneumoniae isolates at the hospital. The association of the enzyme KPC with other resistance determinants, such as loss of porins, may increase the severity of the situation of nosocomial infections. There is an urgent need to develop strategies for infection control and prevention.

[1]  L. Schouls,et al.  The Carbapenem Inactivation Method (CIM), a Simple and Low-Cost Alternative for the Carba NP Test to Assess Phenotypic Carbapenemase Activity in Gram-Negative Rods , 2015, PloS one.

[2]  Shan-Chwen Chang,et al.  Carbapenem-Nonsusceptible Enterobacteriaceae in Taiwan , 2015, PloS one.

[3]  Christopher A. Broberg,et al.  Klebsiella: a long way to go towards understanding this enigmatic jet-setter , 2014, F1000prime reports.

[4]  Alessandra Carattoli,et al.  Plasmids and the spread of resistance. , 2013, International journal of medical microbiology : IJMM.

[5]  A. Marra,et al.  A hospital-based matched case–control study to identify clinical outcome and risk factors associated with carbapenem-resistant Klebsiella pneumoniae infection , 2013, BMC Infectious Diseases.

[6]  F. Rossi,et al.  The modified Hodge test is a useful tool for ruling out klebsiella pneumoniae carbapenemase , 2012, Clinics.

[7]  S. Pournaras,et al.  Predictors of mortality in patients with bloodstream infections caused by KPC-producing Klebsiella pneumoniae and impact of appropriate antimicrobial treatment. , 2011, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[8]  M. Barbier,et al.  OmpK26, a Novel Porin Associated with Carbapenem Resistance in Klebsiella pneumoniae , 2011, Antimicrobial Agents and Chemotherapy.

[9]  F. Baquero,et al.  Dissemination of blaKPC-2 by the Spread of Klebsiella pneumoniae Clonal Complex 258 Clones (ST258, ST11, ST437) and Plasmids (IncFII, IncN, IncL/M) among Enterobacteriaceae Species in Brazil , 2011, Antimicrobial Agents and Chemotherapy.

[10]  D. Sofianou,et al.  Hospital outbreak caused by Klebsiella pneumoniae producing KPC-2 beta-lactamase resistant to colistin. , 2010, The Journal of hospital infection.

[11]  D. Hospenthal,et al.  Impact of extended spectrum beta-lactamase producing Klebsiella pneumoniae infections in severely burned patients. , 2010, Journal of the American College of Surgeons.

[12]  J. Quinn,et al.  Worldwide Diversity of Klebsiella pneumoniae That Produce β-Lactamase blaKPC-2 Gene , 2010, Emerging infectious diseases.

[13]  S. Pournaras,et al.  A simple phenotypic method for the differentiation of metallo-beta-lactamases and class A KPC carbapenemases in Enterobacteriaceae clinical isolates. , 2010, The Journal of antimicrobial chemotherapy.

[14]  Qiwen Yang,et al.  Phenotypic and Genotypic Characterization of Enterobacteriaceae with Decreased Susceptibility to Carbapenems: Results from Large Hospital-Based Surveillance Studies in China , 2009, Antimicrobial Agents and Chemotherapy.

[15]  D. Landman,et al.  Contribution of OmpK36 to carbapenem susceptibility in KPC-producing Klebsiella pneumoniae. , 2009, Journal of medical microbiology.

[16]  G. Peirano,et al.  First Report of KPC-2-Producing Klebsiella pneumoniae Strains in Brazil , 2008, Antimicrobial Agents and Chemotherapy.

[17]  Margaret A Dudeck,et al.  CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting. , 2008, American journal of infection control.

[18]  Gabriela C. Moraes,et al.  Metallo-β-Lactamase Detection: Comparative Evaluation of Double-Disk Synergy versus Combined Disk Tests for IMP-, GIM-, SIM-, SPM-, or VIM-Producing Isolates , 2008, Journal of Clinical Microbiology.

[19]  N. Høiby,et al.  Class A carbapenemases. , 2007, The Journal of antimicrobial chemotherapy.

[20]  D. Landman,et al.  Evolution of antimicrobial resistance among Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae in Brooklyn, NY. , 2007, The Journal of antimicrobial chemotherapy.

[21]  K. Bush,et al.  Carbapenemases: the Versatile β-Lactamases , 2007, Clinical Microbiology Reviews.

[22]  N. Woodford,et al.  Multiplex PCR for rapid detection of genes encoding acquired metallo-beta-lactamases. , 2006, The Journal of antimicrobial chemotherapy.

[23]  D. Paterson,et al.  KPC Type β-Lactamase, Rural Pennsylvania , 2006, Emerging infectious diseases.

[24]  Roberta B. Carey,et al.  Carbapenem Resistance in Klebsiella pneumoniae Not Detected by Automated Susceptibility Testing , 2006, Emerging infectious diseases.

[25]  H. Goossens,et al.  Prevalence and antimicrobial susceptibility data for extended-spectrum beta-lactamase- and AmpC-producing Enterobacteriaceae from the MYSTIC Program in Europe and the United States (1997-2004). , 2005, Diagnostic microbiology and infectious disease.

[26]  A. Maniatis,et al.  Outbreaks in Distinct Regions Due to a Single Klebsiella pneumoniae Clone Carrying a blaVIM-1 Metallo-β-Lactamase Gene , 2005, Journal of Clinical Microbiology.

[27]  D. Landman,et al.  Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular epidemiology and in vitro activity of polymyxin B and other agents. , 2005, The Journal of antimicrobial chemotherapy.

[28]  P. Bradford,et al.  Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 beta-lactamases in New York City. , 2004, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[29]  P. Nordmann,et al.  Emergence of Oxacillinase-Mediated Resistance to Imipenem in Klebsiella pneumoniae , 2004, Antimicrobial Agents and Chemotherapy.

[30]  V. Miriagou,et al.  Imipenem Resistance in a Salmonella Clinical Strain Due to Plasmid-Mediated Class A Carbapenemase KPC-2 , 2003, Antimicrobial Agents and Chemotherapy.

[31]  E. S. Moland,et al.  Occurrence of Newer β-Lactamases in Klebsiella pneumoniae Isolates from 24 U.S. Hospitals , 2002, Antimicrobial Agents and Chemotherapy.

[32]  R. Cantón,et al.  Variations in the prevalence of strains expressing an extended-spectrum beta-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific region. , 2001, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[33]  D. Yong,et al.  Modified Hodge and EDTA-disk synergy tests to screen metallo-beta-lactamase-producing strains of Pseudomonas and Acinetobacter species. , 2001, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[34]  R. Podschun,et al.  Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors , 1998, Clinical Microbiology Reviews.

[35]  L. Chui,et al.  A standardized protocol for the rapid preparation of bacterial DNA for pulsed-field gel electrophoresis. , 1998, Diagnostic microbiology and infectious disease.

[36]  H. Nikaido,et al.  Prevention of drug access to bacterial targets: permeability barriers and active efflux. , 1994, Science.

[37]  J. Quinn,et al.  Selective imipenem resistance in Pseudomonas aeruginosa associated with diminished outer membrane permeability , 1988, Antimicrobial Agents and Chemotherapy.

[38]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[39]  N. Woodford,et al.  Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. , 2006, The Journal of antimicrobial chemotherapy.