Characterization of TEM-1 β-Lactamase-Producing Kingella kingae Clinical Isolates

Abstract

Kingella kingae is a human pathogen that causes pediatric osteoarticular infections and infective endocarditis in children and adults. The bacterium is usually susceptible to β-lactam antibiotics, although β-lactam resistance has been reported in rare isolates. This study was conducted to identify β-lactam-resistant strains and to characterize the resistance mechanism. Screening of a set of 90 K. kingae clinical isolates obtained from different geographic locations revealed high-level resistance to penicillins among 25% of the strains isolated from Minnesota and Iceland. These strains produced TEM-1 β-lactamase and were shown to contain additional ≥50-kb plasmids. Ion Torrent sequencing of extrachromosomal DNA from a β-lactamase-producing strain confirmed the plasmid location of the bla_TEM gene. An identical plasmid pattern was demonstrated by multiplex PCR in all β-lactamase producers. The porin gene's fragments were analyzed to investigate the relatedness of bacterial strains. Phylogenetic analysis revealed 27 single-nucleotide polymorphisms (SNPs) in the por gene fragment, resulting in two major clusters with 11 allele types forming bacterial-strain subclusters. β-Lactamase producers were grouped together based on por genotyping. Our results suggest that the β-lactamase-producing strains likely originate from a single plasmid-bearing K. kingae isolate that traveled from Europe to the United States, or vice versa. This study highlights the prevalence of penicillin resistance among K. kingae strains in some regions and emphasizes the importance of surveillance for antibiotic resistance of the pathogen.

Fig 1

Plasmid DNA analysis in K. kingae strains. (A to C) The bla_TEM gene amplified from K. kingae DNA (A), plasmid DNA identified in K. kingae strains (B), and multiplex PCR plasmid typing (C). Lanes: 1, PYKK081 (Israel; 1991); 2, C2005004457 (United States; 2005); 3, C2007000490 (United States; 2007); 4, M2004000037 (United States; 2004); 5, 0303 and 28260 (Iceland; 2003); 6, 0405 and 30002 (Iceland; 2004); 7, 9508 and 31135 (Iceland; 1995). (D) Plasmid DNA purified using a cesium chloride gradient. Lanes: 1, supercoiled DNA ladder (Invitrogen); 2, plasmid DNA purified from strain C2005004457.

Fig 2

Phylogeny of Kingella strains based on the 956-bp por gene sequence. Strains that produce β-lactamase are underlined. Strains isolated from outbreak patients in Minnesota are indicated with asterisks. The numbers on the right indicate the 11 allele types discussed in the text. The numbers within the dendrogram indicate the occurrence (percent) of the branching in 100 bootstrapped trees. K. oralis por was used as the outgroup.