Emergence of Phytobacter diazotrophicus carrying an IncA/C2 plasmid harboring bla NDM-1 in Tokyo, Japan

Abstract

Phytobacter diazotrophicus is an Enterobacterales species that was originally identified as a plant growth-promoting, Gram-negative bacterium. Recently, this species has been recognized as relevant to opportunistic human and nosocomial infections in clinical settings. Its frequent misidentification as other Enterobacterales species from clinical examination occasionally causes a delay in the identification of nosocomial outbreaks. Here, we report the emergence of New Delhi metallo-β-lactamase (NDM)-producing P. diazotrophicus isolated from hospitalized pediatric patients and hospital environments in Tokyo, Japan. In our case, these isolates were found during an investigation of carbapenem-resistant Enterobacterales in relation to nosocomial infections. Whole-genome sequencing is useful for overcoming the difficulty of species identification. Furthermore, we found that bla _NDM-1 was carried by an IncA/C₂ plasmid (approximately 170 kbp), which was transferrable from the clinical isolates to the recipient strain Escherichia coli J53. Our study demonstrated that P. diazotrophicus behaves as a carrier of bla _NDM-harboring plasmids, potentially disseminating resistance to carbapenems among Enterobacterales. IMPORTANCE Early detection of nosocomial outbreaks is important to minimize the spread of bacteria. When an outbreak is caused by multidrug-resistant bacteria such as carbapenem-resistant Enterobacterales, a delay in findings makes it difficult to control it because such bacteria often spread not only among human patients but also in hospital environments. Phytobacter diazotrophicus, an Enterobacterales species that has recently been found to be relevant to clinical settings, is often misidentified as other bacteria in clinical laboratories. Here, we found NDM-producing P. diazotrophicus in hospitalized pediatric patients and their environment in Tokyo, Japan. Given that the isolates carried bla _NDM-1-harboring transferrable plasmids, the influence of such bacteria could be greater with the mediation of horizontal transfer of carbapenem resistance. Our findings suggest that P. diazotrophicus should be recognized as an NDM-carrier, for which more attention should be paid in clinical settings.

Keywords: Enterobacteriaceae; antibiotic resistance; genome analysis; genotypic identification; molecular epidemiology; plasmid-mediated resistance.

Fig 1

Phylogenetic trees based on whole-genome data. Clinical isolates were compared to reference strains based on ANI (left) and core genes (right). These trees were constructed using the neighbor-joining method. Clinical isolates and P. diazotrophicus references are shown by the elliptical area highlighted in cyan. Metakosakonia sp. MRY16-398 and Citrobacter sp. BDA59-3 are regarded as P. diazotrophicus MRY16-398 and P. diazotrophicus BDA59-3, respectively, and the reclassification of Metakosakonia massiliensis to Phytobacter massiliensis is included, according to recent studies (10, 21). Scale bars = distance.

Fig 2

Circular map of the pTMTA97342 plasmid. IncA/C₂ plasmids were compared and visualized using the GView Server (27). Alignment length and percentage of identity cutoff values for sequence-based BLASTn analysis revealed that the colored bars for these plasmids were 80% and 100%, respectively. The positions of open reading frames were derived from those in pTMTA98742, except for repA, which was derived from pNDM-TAEC1.