Genotypic comparison of Pantoea agglomeransplant and clinical strains

Abstract

BackgroundPantoea agglomerans strains are among the most promising biocontrol agents for a variety of bacterial and fungal plant diseases, particularly fire blight of apple and pear. However, commercial registration of P. agglomerans biocontrol products is hampered because this species is currently listed as a biosafety level 2 (BL2) organism due to clinical reports as an opportunistic human pathogen. This study compares plant-origin and clinical strains in a search for discriminating genotypic/phenotypic markers using multi-locus phylogenetic analysis and fluorescent amplified fragment length polymorphisms (fAFLP) fingerprinting.ResultsMajority of the clinical isolates from culture collections were found to be improperly designated as P. agglomerans after sequence analysis. The frequent taxonomic rearrangements underwent by the Enterobacter agglomerans/Erwinia herbicola complex may be a major problem in assessing clinical associations within P. agglomerans. In the P. agglomerans sensu stricto (in the stricter sense) group, there was no discrete clustering of clinical/biocontrol strains and no marker was identified that was uniquely associated to clinical strains. A putative biocontrol-specific fAFLP marker was identified only in biocontrol strains. The partial ORF located in this band corresponded to an ABC transporter that was found in all P. agglomerans strains.ConclusionTaxonomic mischaracterization was identified as a major problem with P. agglomerans, and current techniques removed a majority of clinical strains from this species. Although clear discrimination between P. agglomerans plant and clinical strains was not obtained with phylogenetic analysis, a single marker characteristic of biocontrol strains was identified which may be of use in strain biosafety determinations. In addition, the lack of Koch's postulate fulfilment, rare retention of clinical strains for subsequent confirmation, and the polymicrobial nature of P. agglomerans clinical reports should be considered in biosafety assessment of beneficial strains in this species.

Application of Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Identification and Clustering Analysis ofPantoeaSpecies

“…A collection of 53 strains received as P. agglomerans, a Pantoea sp., or E. agglomerans from research and culture collections and 20 reference strains belonging to closely related Pantoea species and other Enterobacteriaceae were compared ( Table 1). The streptomycin-resistant, commercial biocontrol strain Pantoea vagans C9-1S (formerly P. agglomerans [45]) and a variant strain, P. vagans C9-1W, lacking the 530-kb pPag3 plasmid (49), were included to evaluate the sensitivity and robustness of MALDI-TOF MS. For standardized spectral acquisition and generation of "SuperSpectra," as determined by SARAMIS (spectral archive and microbial identification system) (AnagnosTec GmbH), bacteria were grown on LB agar at 28°C for 24 to 48 h. Tryptic soy agar (TSA) and Mueller-Hinton agar (MHA) were used in parallel to assess the influence of different media on species recognition. Alongside MALDI-TOF MS analysis, biochemical profiling and conventional Sanger sequencing of the gyrB gene were performed for all strains.…”

“…Amplification and sequencing of the gyrB gene were performed as described previously (45) by means of the HotStarTaq master mix kit (Qiagen, Basel, Switzerland) and the ABI PRISM BigDye Terminators version 1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA), respectively. Two degenerate primers were used to amplify and sequence a 970-bp region of the gyrB gene, gyr-320 (5Ј-TAARTTYGAYGAYAACTCYTAYAAAGT-3Ј) and rgyr-1260 (5Ј-CMCCYTCCACCARGTAMAGTTC-3Ј) (15).…”

“…Accurate identification is complicated by the unsettled taxonomy of the "P. agglomerans-E. herbicola-E. agglomerans" complex (45). Recent analyses based on gyrB sequencing, multilocus sequence analysis (MLSA) (4), and fluorescent amplified fragment length polymorphisms (fAFLP) (45) indicate that strains belonging to Enterobacter or Erwinia archived in culture collections are often erroneously assigned to P. agglomerans and are likely also misidentified in clinical diagnostics. False classifications of environmental P. agglomerans strains as related Pantoea species, including human-or plant-pathogenic P. ananatis, are also common (45).…”

“…Recent analyses based on gyrB sequencing, multilocus sequence analysis (MLSA) (4), and fluorescent amplified fragment length polymorphisms (fAFLP) (45) indicate that strains belonging to Enterobacter or Erwinia archived in culture collections are often erroneously assigned to P. agglomerans and are likely also misidentified in clinical diagnostics. False classifications of environmental P. agglomerans strains as related Pantoea species, including human-or plant-pathogenic P. ananatis, are also common (45). Inadequate biochemical identification methods and uncertainty regarding current taxonomy are revealed also by the excessive number of 16S rRNA gene sequences incorrectly assigned to P. agglomerans that can be retrieved from GenBank ( Fig.…”

“…While several strains are commercialized for biological control of plant diseases (23), the species also includes two phytopathogenic pathovars that carry distinctive virulence plasmids (32). P. agglomerans has a Jekyll-Hyde nature, being described also as an opportunistic human pathogen (30), which raises biosafety regulatory issues for the utilization of beneficial isolates (45). Clinical reports predominantly involve septicemia following penetrating trauma (16,56) or nosocomial infections (14,55).…”