Penicillinase-Encoding Gene bla_TEM-1 May Be Plasmid Borne or Chromosomally Located in Kingella kingae Species

LETTER

Kingella kingae is the prime pathogen of osteoarticular infections in young children in numerous countries (1–4). Although K. kingae is usually susceptible to antibiotics that are administered to children with skeletal system infections, β-lactamase production has been sporadically detected in Iceland (5), the United States (6), Israel (7), and, more recently, in France (8). Using genotyping methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), β-lactamase production appeared as clonally distributed. Indeed, four different clones have been described in Israel (PFGE clone A/sequence type complex 34 [STc-34], clone F/STc-3, clone Ψ/ST-40, and clone T/ST-not typeable), one common clone was isolated in Iceland and the United States (clone K/ST-6), and the French isolate belonged to STc-14 (8, 9). Although the U.S. and Icelandic β-lactamase producers were associated with a bla_TEM-1 gene carried by a conjugative plasmid, the French isolate possesses a chromosomally located bla_TEM-1 gene (8, 10).

In this report, we investigated the bla_TEM-1 gene location in randomly chosen strains belonging to the four Israeli β-lactamase-producing clones, using PFGE of I-CeuI-digested DNA, followed by hybridization on a Southern blot with bla_TEM-1 and 16S/23S rRNA gene probes, as previously described (8). The Icelandic 0211 + 12480 and U.S. C2005004457 strains were used as plasmid-borne β-lactamase-producing controls, while the French KWG1 strain was used as a chromosome-encoded β-lactamase-producing control.

The observed colocalization of 16S/23S rRNA and bla_TEM-1 genes demonstrated that the strains belonging to clone A/STc-34 but to two different STs (KK245, belonging to ST-33, and BB114, belonging to ST-34) possessed a chromosomally located bla_TEM-1 gene (Fig. 1). In contrast, in strains belonging to clone F/STc-3 (AA046, belonging to ST-3, and KK545, belonging to ST-39) and that belonging to clone Ψ/ST-40 (strain 15/4.267), the bla_TEM-1 probe hybridized elsewhere out of the chromosome, indicating a plasmid-borne gene (Fig. 1). Of note, strains belonging to clones F and Ψ presented at least 3 bands hybridizing with the bla_TEM-1 gene probe. Whether these bands correspond to multiple plasmids or different plasmid conformations remains to be determined. Surprisingly, our results may suggest that the clone T/ST-not typeable strain (D2756) harbored a double location of the bla_TEM-1 gene, plasmidic as well as chromosomal. Indeed, one band hybridized with both probes and another one hybridized only with the bla_TEM-1 probe (Fig. 1). However, in contrast to this strain, all the other chromosomal locations are on a very heavy DNA band and had a weak hybridization of the bla_TEM-1 probe. This can suggest that these two bands may be from plasmids. Nevertheless, we previously described, by 16S rRNA gene sequencing and MLST, that the strains belonging to clone T may represent a K. kingae subspecies (9), which could explain a different chromosomal organization.

FIG 1.

Composite figure showing pulsed-field gel electrophoresis of I-CeuI-digested DNA (A) and Southern blot analysis using the bla_TEM-1 gene probe (B) and a 16S/23S rRNA gene probe (C) of Kingella kingae penicillinase-producing strains. λ, molecular marker; lanes 1 (KK245) and 2 (BB114), Israeli clone A strains; lanes 3 (AA046) and 4 (AA545), Israeli clone F strains; lane 5 (154.267), Israeli clone Ψ strain; lane 6 (D2756), Israeli clone T strain; lanes 7 (0211 + 12480) and 8 (C2005004457), Icelandic and U.S. plasmid-borne β-lactamase producers belonging to clone K, respectively; lanes 9 (KWG1), French chromosome-encoded β-lactamase producer belonging to sequence type complex 14. The black arrows show the chromosomal bands hybridizing with the bla_TEM-1 probe, while the white arrows show locations hybridizing with the bla_TEM-1 probe out of the chromosome.

The present results demonstrate that chromosomal location of the bla_TEM-1 gene is not restricted to the French K. kingae strain but also is present at least in one different clone in Israel (clone A). These results indicate that several events of bla_TEM-1 gene integration have occurred independently in the species, since these different clones are only distantly related (9, 11). To our knowledge, bla_TEM-1 gene integration into the chromosome has not been previously described in the Neisseriaceae family, and particularly in closely related species such as Neisseria gonorrhoeae. Whether K. kingae genetic determinants exist to promote this phenomenon remains to be determined.

In previous studies, we observed that clone A strains, as well as the French isolate, exhibited a lower amoxicillin MIC than that of clones F and K (8, 9). This result may suggest that the chromosomally located bla_TEM-1 gene confers a low-level resistance to penicillin drugs compared to the plasmid-borne gene. Although the reason for this phenomenon is still unknown, it could indicate that production of the enzyme is controlled by regulatory mechanisms or that plasmids are present in multicopy and then produce more TEM-1 enzyme. Microbiologists should be aware of this low level of resistance in invasive clinical isolates of K. kingae, which should be routinely tested for the presence of β-lactamase using the chromogenic Cefinase test.