Low prevalence of rmpA and high tendency of rmpA mutation correspond to low virulence of extended spectrum β-lactamase-producing Klebsiella pneumoniae isolates

Abstract

Invasive syndrome caused by Klebsiella pneumoniae (KP), including liver abscess, is mainly caused by community-acquired strains with characteristics of positive hypermucoviscosity (HV) phenotype and regulator of mucoid phenotype A (rmpA) and transcriptional activator (rmpA2) genes. Extended- spectrum β-lactamase-producing KP (ESBL-KP) is commonly nosocomial and rarely HV-positive. We aimed to explore the reasons of the rarer prevalence of HV phenotype, rmpA and rmpA2 as well as the virulence phenotype among the ESBL-KP isolates from clinical specimens than those non-ESBL isolates. The β-lactamase genes, rmpA, rmpA2 and genes for K capsule serotype of 440 KP isolates were analyzed. The virulence of the isolates was characterized by the mouse lethality experiments. The prevalence rates of HV phenotype (∼ 50% vs. < 10%) as well as rmpA and rmpA2 genes (∼ 50-60% vs. < 20-30%) were significantly higher in non-ESBL group than in the ESBL group (p < 0.0001). Expression of HV phenotype in the rmpA-positive KP isolates was significantly rarer in the ESBL group than in non-ESBL group (33.3% vs. 91.9%, p < 0.0001). The frameshift mutations of rmpA and/or rmpA2 corresponded to negative HV phenotype of KP isolates that harbored the rmpA and/or rmpA2, resulting in variable mouse lethality (LD50, ∼ 10(3) - >5 × 10(7) CFU). The mutation rates might significantly differ among KP isolates from various sources. Virulence was dependent on rmpA-related HV phenotype. In conclusion, ESBL-KP isolates were less hypermucoviscous and less virulent than non-ESBL KP isolates, mostly due to concurrently lower carriage and higher mutation rates of the rmpA and rmpA2 genes.

Keywords: ESBL hypermucoviscosity; klebsiella pneumoniae; rmpA gene; spontaneous mutation.

Figure 1.

Alignment analysis of rmpA sequences. Multiple sequences are compared with the reference sequence of GenBank accession number AB289642, which defined plasmid pK2044 rmpA gene from the strain K. pneumoniae NTUH-K2044 (http://www.ncbi.nlm.nih.gov/nuccore/AB289642). The strains KP 309, 401, 420, and 425 represent isolates which were PCR-positive for rmpA and rmpA2 but negative for hypermucoviscosity phenotype. The strain KP291 represents isolates without hypermucoviscosity phenotype, which were PCR-positive for rmpA but PCR-negative for rmpA2. The strain KP449 represents isolates with hypermucoviscosity phenotype, which were PCR-positive for rmpA and rmpA2.

Figure 2.

Alignment analysis of rmpA2 sequences. Multiple sequences are compared with reference sequences of GenBank accession numbers S64176 and AB289643. The S64176 defined plasmid rmpA2 gene from the strain K. pneumoniae, Chedid, O1:K2 serotype (http://www.ncbi.nlm.nih.gov/nuccore/S64176). The AB289643 revealed a truncated rmpA2 region and a nucleotide deletion in the poly-G tract of ORF KPP302 from the strain K. pneumoniae NTUH-K2044 (http://www.ncbi.nlm.nih.gov/nuccore/AB289643).

Figure 3.

DNA analysis for transconjugational plasmids of transconjugants 290–3, 414–3 and 417–3 by gel electrophoresis (A) without restriction enzyme (B) with restriction enzyme BamHI or Hind III. M1: MidRange Marker II (New England BioLabs, Ipswich, MA, USA); M2:1 kb DNA Ladder (Protech Technology Enterprise Co., Ltd, Taipei, Taiwan). Southern blotting for bla_SHV-5 (C, upper) and rmpA (C, lower) on transconjugational plasmid of transconjugants was shown.

Figure 4.

Surviving curve for each group of 4 mice per inoculum dose of strain KP309 (rmpA/A2 frameshift mutant, non-HV phenotype, K2 capsule stereotype) expressing a LD₅₀ of 2.1 x 10³ CFU.