Phytobacter spp: the emergence of a new genus of healthcare-associated Enterobacterales encoding carbapenemases in Argentina: a case series

Summary Members of the genus Phytobacter (order Enterobacterales) are isolated from the natural environment and clinical settings. Identification of Phytobacter strains based on biochemical characteristics is complicated due to taxonomic confusion, and they are often misidentified by automated identification systems in laboratories. In this study we describe the first three clinical cases associated with Phytobacter spp. reported in Argentina. We describe the identification, the molecular analysis using whole genome sequencing and the potential clinical relevance.


Introduction
Members of the genus Phytobacter (order Enterobacterales) may be isolated from both the natural environment and from clinical settings [1e3].They are recognised as plant growth-promoting rhizobacteria helping plants grow.However, in recent times they are increasingly reported in clinical infections such as bacteraemia such as cases in Brazil resulting from contaminated total parenteral nutrition [4].
Phytobacter diazotrophicus, was originally described by Zhang et al. [2] based on few endophytic nitrogen-fixing bacteria isolated from surface-sterilised stems and roots of wild rice (Oryza rufipogon) in Hainan, China.Later, Pillonetto et al. emended the description of the genus Phytobacter and its type species Phytobacter diazotrophicus and described a new species Phytobacter ursingii sp.nov.[1].
In 2020, two other species were assigned to Phytobacter genus.Ma et al. proposed the unification of the genus Metakosakonia and the genus Phytobacter to a single genus Phytobacter and reclassified Metakosakonia massiliensis as Phytobacter massiliensis comb.nov.[6].Madhaiyan et al. describe Phytobacter palmae, a new species isolated as an endophyte from surface-sterilised leaflet tissues of oil palm (Elaeis guineensis) from Singapore [7].
The identification of Phytobacter strains based on biochemical characteristics is complicated due to taxonomic confusion, and they are often misidentified by automated identification systems in laboratories [1].
Smits et al. have further unravelled the taxonomic confusion of the genus Phytobacter using modern molecular tools based on whole-genome sequencing analysis with digital DNA-DNA hybridization and average nucleotide identities (ANI), assigning isolates previously characterised as Pantoea agglomerans into two distinct species: Phytobacter diazotrophicus and Phytobacter ursingii [3].
In the present study we describe the first three clinical cases in Argentina in which Phytobacter spp.were isolated from clinical samples.

Case 1
An 85-year-old female with breast cancer and lung metastasis undergoing chemotherapy was admitted with symptoms of fever, syncopal episode, dyspnoea, and a productive cough.She had a history of previous hospitalisations.The clinical interpretation of her presentation was that the symptoms were likely to represent hospital-acquired pneumonia.She was treated with piperacillin-tazobactam for 7 days and had a good clinical response.
From a rectal swab, which had been sent for screening for carbapenemase-producing Entrobacterales (CPE), an Enterobacterium grew which was identified by whole genome sequencing (WGS) as Phytobacter diazotrophicus.Three acquired resistance genes were detected in this organism: bla SHV-12 , bla KPC-2 , located in a transposon (Tn4401) and bla qnrE , responsible for resistance to quinolones.

Case 2
A 26-day-old female premature neonate with a birth weight of 1000g, had been born following an extra-membranous pregnancy which occurred from week 19 of the pregnancy.She had required advanced cardiopulmonary resuscitation (endotracheal tube placement, chest compressions, and administration of adrenaline) and mechanical respiratory assistance for 14 days.She had also been treated with UV light therapy for 3 days due to neonatal jaundice.At birth, blood cultures were collected which subsequently had no growth.She completed antibiotic treatment with intravenous ampicillin for 7 days and gentamicin for two days, because of slow clinical progress.She had received parenteral nutrition since the first day of life for 14 days.On the 13th day following birth, she decompensated clinically and haemodynamically.Blood cultures were collected (2 samples; BACTECÒ Peds Plus Medium).Gram-negative bacilli subsequently identified as Phytobacter ursingii resistant to ampicillin only, grew in both samples after 7 hours' incubation.She had received further treatment with ampicillin and gentamicin but did not respond to treatment and died.

Case 3
A 78-year-old man patient, with a history of benign prostatic hyperplasia, presented with fever and chills 10 days after a prostatectomy.Urine and blood cultures were collected.The urine analysis showed 2e4 leukocytes/HPF, >20 erythrocyte/ HPF, 0e1 epithelial cells/HPF.
Phytobacter diazotrophicus identified by WGS and which was resistant to ampicillin was isolated from the urine (with a colony count greater than 100,000 CFU (colony forming units)/ ml and from the blood cultures.The BACTECÒ Lytic/10 Anaerobic and BACTEC Plus Aerobic blood cultures were positive, after 18 and 28 hours' incubation respectively.The clinical presentation was interpreted as sepsis with a likely urinary source.The patient was treated with piperacillin-tazobactam and ciprofloxacin with a good clinical response.In all three cases, the colonies on nutrient agar were nonpigmented and on EMB Levine Agar (Difco Ô) the organisms produced mucoid lactose-fermenting colonies (Figure 1).
The phenotypic identification of the isolates was initially carried out with conventional biochemical tests.Using this methodology, identification of the 3 isolates was compatible with non-pigmented, indole producing Pantoea spp.(Table I) Identification by the use of an automated Vitek 2 Ò compact system (bioMe ´rieux,Marcy-l'E ´toile, France) using a GN Colorimetric Identification Card yielded respectively, the following biocodes: 601714453500010 (Unidentified); 607730457500000 (Pantoea spp.95 %), and 607734557500050 (Pantoea spp.89 %).
To confirm the identification of our isolates, WGS was performed using the Illumina MiSeq sequencer at the National Center for Genomics and Bioinformatics ANLIS "Dr Carlos G Malbra ´n", Buenos Aires, Argentina.The assembly of the sequence was carried out using SPAdes Assembler (version.3.1.0).Whole genomes were deposited at DDBJ/EMBL/GenBank under the accession no.JAWJAA000000000 (P.diazotrophicus CVMA1), JAWJAB000000000 (P.diazotrophicus CVMA24) and JAW-JAC000000000 (P.ursingii CVMA36).
The sequenced genomes were annotated with the Prokka [8] and Roary software [9] was used to align the core genes.The SNP-sites program was used to identify polymorphisms (SNPs) [10], to then carry out the phylogenetic study using IQTREE2 under the GTR model with the gamma distribution (GTRþGAMMA) predicted by ModelFinder [11].To carry out the analysis of the genomes, various bioinformatics tools were used.Species identification by genome sequence were performed by ANI% and DNA-DNA hybridization in silico using ani.rbscript [12] and GGDC software [13], respectively.Likewise, the determination of the resistome and virulome was carried out with BLAST (E-value<10 À5 ) and different databases such as MegaRes [14] and VFDB database [15].
Antimicrobial resistance gene identification was conducted using the MegaRes database.Phytobacter diazotrophicus CVMA1 contains the bla KPC-2 , bla SHV-12 genes (harbored within the Tn4401 transposon), and qnrE1 genes, responsible for resistance to quinolones.We also identified a homologous gene to fosA (Amino Acid Identity: 75.31%;Coverage: 95.95%) in all the sequenced isolates, which could explain the fosfomycin resistance phenotype.Virulence gene identification was carried out using the VFDB database.In Phytobacter genomes, 68, 69, and 70 virulence genes were identified in CVMA1, CVMA24, and CVMA36, respectively (Supplementary Table S1).
The antibiotic susceptibility tests were performed using the VITEKÒ 2 system with the AST-079 (GNS susceptibility card) panel.The MIC breakpoints used in this study were those established by the Clinical and Laboratory Standards Institute [16] for Enterobacterales (Table II).In addition, fosfomycin susceptibility was determined using a 200 mg disk test in agar Mueller Hinton.CMVA1 and CMVA 24 showed no halo (6mm) and CMVA36 showed a small halo (15mm), therefore all the isolates were resistant.

Discussion
The colonial morphology of Phytobacter spp. on EMB Levine agar, resembles E. coli and Citrobacter since it is a strong lactose-fermenting Gram-negative bacillus.However, it can also be lactose non-fermenting and have a colonial morphology that resembles Enterobacter cloacae complex.Given that the biochemical tests for Phytobacter spp include negative results for lysine decarboxylase, ornithine decarboxylase and arginine dihydrolase, it can be misidentified as Pantoea spp. or Pantoea agglomerans by means of conventional biochemical tests or, by automated identification systems, as has been previously pointed out by other authors [4,17] potentially resulting in the overestimation of the role of this latter species in human infection [4,18].
Likewise, the identification by means of mass spectrometry systems (including MALDI-TOF MS, VitekÒ-MS and MALDI Biotyper-Bruker MicroflexÒ) can also be incorrect if the database is not updated [2].
Although Phytobacter has been described in human samples by Pillonetto M et al. [1] only recently whole genome sequencing has shown that the genus Phytobacter has been incorrectly classified as other species since the 1970s.[3].
Its clinical relevance has been demonstrated in studies, including 24 isolates studied by Smits et al. [3] in a period of 5 years (2016e2021) with more than 50% recovered from blood cultures.It has also been implicated in outbreaks worldwide including in Brazil and the United States.[4].
Many of the cases identified as having Phytobacter spp infection were sepsis after receiving intravenous fluids or the use of medical devices, often in neonatal intensive care units [4].
The finding of multi-resistant strains of Phytobacter spp has been reported by other authors [2,12,19,20].One of our isolates was multi-resistant (with 2 acquired resistance genes for beta-lactam antibiotics: bla SHV -12 , and bla KPC-2 , located in a transposon (Tn4401)), demonstrating its ability to become a reservoir of resistance genes in the hospital environment.Every isolate showed resistance to fosfomycin.The phenotype could be explained by the presence of a homologous gene of fosA.

Conclusions
Although there are only several reported cases of Phytobacter infections, its true clinical relevance could be hidden due to the phenotypic misidentification and the lack of reference organisms in MALDI-TOF MS databases.The emergence of new genera of Enterobacterales has posed challenges for the diagnosis and treatment of related infections.In this sense, since sequencing approaches might not be available for many laboratories, the correct identification of Phytobacter spp.remains a challenge for routine clinical microbiology diagnostics.actively in the interpretation of the microbiological results and approved the final version of the manuscript.

Figure 1 .
Figure 1.Colonies of a Phytobacter isolate on EMB Levine.