Detection of bla KPC-2 in Proteus mirabilis in Brazil

Abstract

INTRODUCTION

: Infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing isolates pose a major worldwide public health problem today.

METHODS

: A carbapenem-resistant Proteus mirabilis clinical isolate was investigated for plasmid profiles and the occurrence of β-lactamase genes.

RESULTS

: The isolate exhibited resistance to ertapenem and imipenem and was susceptible to meropenem, polymyxin, and tigecycline. Five plasmids were identified in this isolate. DNA sequencing analysis revealed the presence of bla KPC-2 and bla TEM-1 genes. An additional PCR using plasmid DNA confirmed that bla KPC-2 was present in one of these plasmids.

Conclusions:

We report the detection of bla KPC-2 in P. mirabilis in Brazil for the first time. This finding highlights the continuous transfer of bla KPC between bacterial genera, which presents a serious challenge to the prevention of infection by multidrug-resistant bacteria.

Klebsiella pneumoniae carbapenemase (KPC)-type enzymes were first reported in carbapenem-resistant K. pneumoniae strains in North Carolina, United States(1). Until 2005, the geographical distribution of these enzymes in members of the family Enterobacteriaceae - K. pneumoniae, in particular - was limited to the eastern part of the United States. In Brazil, KPC has been described since 2006, and it was first reported in K. pneumoniae isolates from Recife(2), followed by reports(3) in other species from the Enterobacteriaceae and non-fermenting bacilli(4) (5) (6). Carbapenem resistance in Proteus mirabilis caused by KPC-2 was also first reported in the United States(7) in 2008, and currently, there are a few reports of P. mirabilis KPC producers worldwide(8) (9). The present report describes the detection of bla KPC-2 in P. mirabilis strains in Brazil for the first time.

In May 2012, an isolate of Proteus mirabilis was recovered from the blood culture of a patient admitted to the intensive care unit of a tertiary hospital located in Recife, Pernambuco, Brazil. Biochemical identification and determination of minimum inhibitory concentrations (MICs) of the isolate were performed using the BD PhoenixTM Automated Microbiology System, Franklin Lakes, New Jersey, USA and the susceptibility profile was determined according to the guidelines of the Clinical and Laboratory Standards Institute (2013). The isolate was characterized as multidrug resistant, showing resistance to amikacin (MIC>32µg/mL), amoxicillin/clavulanate (MIC>16µg/mL), ampicillin (MIC>16µg/mL), cefazolin (MIC>16µg/mL), cefepime (MIC>16µg/mL), cefotaxime (MIC>32µg/mL), cefoxitin (MIC=16µg/mL), ceftriaxone (MIC>32µg/mL), ciprofloxacin (MIC>2µg/mL), ertapenem (MIC>4µg/mL), imipenem (MIC>8µg/mL), gentamicin (MIC>8µg/mL), levofloxacin (MIC>4µg/mL), piperacillin/tazobactam (MIC>64µg/mL), and tobramycin (MIC>8µg/mL). It was susceptible only to meropenem, polymyxin, and tigecycline. Plasmid DNA was extracted by the UltraClean Endotoxin-Free Mini Plasmid Prep Kit (Mo Bio Lab, Carlsbad, CA, USA) and was visualized and analyzed by electrophoresis on a 0.7% agarose gel. The molecular weight of each plasmid was determined by comparison with plasmids of known molecular weight, e.g., with that of K. pneumoniae K16-P strain(3). Five plasmids with estimated molecular sizes of >150kb, 150kb, 120kb, 90kb, and 70kb were identified in the isolate. Genes bla KPC, bla VIM, bla IMP, bla SPM, bla GES, bla SHV, bla TEM, and bla CTX-M were analyzed by PCR using genomic DNA and specific primers(3). bla VIM, bla IMP, bla SPM, bla GES, bla CTM-M, and bla SHV were not present, while bla KPC and bla TEM were detected, amplifying fragments of approximately 1,000bp . An additional PCR using plasmid DNA for bla KPC detection was performed to confirm the plasmid origin of this gene. The coding regions of bla KPC and bla TEM were sequenced, and the analysis of the nucleotide sequences and deduced protein sequences with BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and EXPASY (http://www.expasy.org translate) showed that the isolate harbored bla KPC-2 and bla TEM-1 (GenBank accession numbers KC736925 and KF811201). bla TEM has been described in P. mirabilis from Brazil by Dropa et al. (10) and Abreu et al. (11), but they did not sequence the gene. The analysis of antimicrobial agents revealed susceptibility to meropenem, polymyxin, and tigecycline. Tibbetts et al. (7) identified KPC-producing Proteus mirabilis isolates that were susceptible only to piperacillin/tazobactam and ciprofloxacin in USA, and Sheng et al. (8) identified a panresistant KPC-producing P. mirabilis isolate in China. According to Castanheira et al. (12), meropenem, ceftazidime-avibactam, and tigecycline were the most active antimicrobials against the KPC-producing Escherichia coli, K. pneumoniae, Klebsiella oxytoca, and P. mirabilis isolates collected in 2012 from the all United States Census Bureau-designated regions. The identification of bla KPC-2 as the underlying basis of carbapenem resistance in P. mirabilis isolates is worrisome, although this finding is not totally unexpected, given the recent documented spread of bla KPC carbapenemase to a number of gram-negative bacteria(4) (5) (13) (14), including in K. pneumoniae and Pseudomonas aeruginosa in the same hospital in Recife, Brazil(3) (6). These data indicate the intra-bacterial transference of bla KPC-2 at this hospital. According to the literature, bla KPC is encoded by transposon Tn4401, found in a variety of transferable plasmids(2) (15). In this study, PCR experiments using plasmid DNA confirmed that bla KPC-2 was present in the plasmids. Considering that transposons are mobile genetic elements, bla KPC-2 could be present in any of the 5 plasmids detected (>150 kb, 150kb, 120kb, 90kb, and 70kb). The data presented herein confirm that P. mirabilis strains harboring bla KPC-2 are emerging in Brazil, suggesting the continuous transfer of bla KPC between bacterial genera. This poses a serious challenge to prevent infections caused by multidrug-resistant bacteria.