First case of bloodstream infection caused by Mixta hanseatica sp. nov., a novel species within the Mixta genus of the Erwiniaceae family

Abstract

Members of the Erwiniaceae family very rarely cause infections in humans. Here we describe the first case of a bloodstream infection due to Mixta hanseatica sp. nov., a novel member of the Erwiniaceae family.

1. Introduction

We here describe the first isolate of Mixta hanseatica sp. nov. isolated from the blood culture of a 98-year-old patient with clinical signs of sepsis. Phylogenetic analysis of the isolate is based on its newly assembled complete genome produced by a hybrid assembly using both long- and short-read sequencing. Furthermore, phenotypic data is presented.

2. Case report and isolate cultivation

In November 2020 a 98-year-old man presented to the emergency department after a fall at home with multiple skin abrasions and signs of systemic infection (CRP of 281 mg/l and leucocytes 18.5/nl). Particularly a wound at the left hip showed signs of cellulitis with reddening and hyperthermia. The patient had a body temperature of 35.6°C, a heart rate of 80 beats per minute and systolic and diastolic blood pressure of 192/102 mm Hg. Antibiotic therapy with ampicillin-sulbactam (3 × 3g) was initiated. The next day CRP and leukocytes had significantly dropped to 164 mg/l and 13.4/nl, respectively. A blood culture drawn from the patient turned positive after 5 hours of incubation and microscopy of the positive blood culture revealed singly located Gram-negative rod-shaped bacilli of 2–3μm length. Subculture on Columbia blood agar in 5% CO₂ showed greyish, non-hemolytic mucoid colonies with depressed centre, firmly attached to the agar. The isolate (internal identification number X22927) was catalase-positive and oxidase negative. MALDI-Tof mass spectrometry fingerprinting (Bruker Daltonics) showed highest spectral similarity to an unclassified Erwinia species with an identification score slightly above 2.0. Therefore, 16S-sequencing was performed (primers 27F: AGA GTT TGA TCM TGG CTC AG and 907R: CCG TCA ATT CMT TTR AGT TT). The sequencing results yielded matches in a GenBank search with Mixta theicola, Pantoea alhagi and Erwinia amylovora with a similarity of 99.88% each (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Thus, 16S-rDNA sequencing and MALDI-Tof results indicated isolate X22927 belonging to the family of Erwiniaceae but did not allow identification of the exact species. Biochemical characterization was carried out using the API 20 E, API 50 CH and the API M medium (Biomerieux). Results are shown in Supplementary Table 1. Susceptibility testing with disc diffusion and Vitek 2 (Biomerieux) utilizing EUCAST breakpoints for Enterobacterales (Version 10, valid from 01 to 12-2020) yielded identical results with resistance to Ampicillin and Piperacillin, but susceptibility to Ampicillin-Sulbactam, Piperacillin-Tazobactam, Ceftriaxon, Ceftazidime, Cefepime, Imipenem, Meropenem, Ciprofloxacin, Levofloxacin, Tobramycin and Trimethoprim/Sulfamethoxazole.

Due to rapidly decreasing CRP, antibiotic therapy was prematurely terminated already after three days and the patient was transferred to a geriatric rehabilitation department. After a few days the CRP started to rise again and without drawing additional blood cultures an additional therapy with ceftriaxone was given for eight further days, which resulted in a sustained drop of the CRP and resolution of the skin lesion. Due to protracted clinical improvement the patient was transferred after a hospital stay of three weeks to a short-term nursery home.

3. Whole genome sequencing and phylogenetic analysis

As 16S-based identification can be challenging in Enterobacterales, whole genome sequencing was performed by Nanopore long-read and Illumina short-read sequencing, integrating confident resolution of complex DNA segments with highly accurate base calling.

The DNA library was prepared as previously described [1] and whole genome sequencing was conducted on a NextSeq 500 system and a 300-cycle mid-output kit (Illumina, San Diego, CA, USA). A total of 4,073,032 151 nucleotides paired-end Illumina reads were generated. Bases less than Q30, as well as adapter sequences of the reads, were trimmed and any reads shorter than 35 nt were removed using Trimmomatic v0.36 [2]. After trimming, the sample contained 3,865,434 high-quality paired-end reads for hybrid assembly.

Additionally, high-molecular weight DNA was extracted with the QIAamp Mini Kit (Qiagen, Hilden, Germany). Long-read Nanopore sequencing was performed according to the manufactures protocol (Oxford Nanopore Technologies, Oxford, UK) on ONT GridIOn X5 R9.4.1 flowcells. Base-calling was done with MinKNOW 4.3 ("Super-accuracy" basecalling model) and Guppy (https://nanoporetech.com/). A total of 77,786 reads were generated. Further filtering removed reads with Q-score <10, leaving 55,537 reads with a mean length of 3.3 kb and a maximal length of 110.7 kb. The resulting high-quality long-reads together with Illumina short-reads were fed into the hybrid assembler Unicycler version 0.4.9 [3], resulting in a genome including a complete and circular chromosome of 4.3Mb and four plasmids (94.1, 37.8, 8.4 and 2.3kb, respectively) with mean coverage depths of 41.2x (long-reads) and 126.2x (short-reads). GC-content was 54.43%. Whole genome-based taxonomic analysis was conducted with Type Strain Genome Server (TYGS)(https://tygs.dsmz.de) [4]. Briefly, the TYGS analysis comprised of automatic determination of the ten most closely related type strains [[5], [6], [7]] plus a user added number of Mixta sp. non-type strains (see Fig. 1 and Supplementary Table 2) and phylogenomic inference based on pairwise comparisons among this set of genomes with calculation of 100 distance replicates each. Digital DNA-DNA-Hybridization (dDDH) values and confidence intervals were calculated using the recommended settings of the GGDC 2.1 [8]. A type-based species clustering using a 70% dDDH radius around each of the strains was done as previously described for species discrimination [4]. In addition, the average nucleotide identity (ANI) was calculated with the ChunLab's online Average Nucleotide Identity (ANI) calculator between X22927 and its closest relative Mixta theicola (GenBank accession number NZ_CP028273) [9,10].

Fig. 1.

Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences [20]. The branch lengths are scaled in terms of GBDP distance formula d5. Numbers above branches represent GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 87.9%. The tree was rooted at the midpoint [21]. Analysis was conducted with the Type Strain Genome Server (TYGS, Leibniz Institute DSMZ).

The phylogenetic analysis revealed 83.23% average nucleotide identity (ANI) and a dDDH value by formula d4 of 39.1% compared to its closest relative Mixta theicola (GenBank accession number NZ_CP028273) [4,9]. Thus, the criteria for a new species with recommended cut-offs of 95–96% and 70 %, respectively, were met and the name Mixta hanseatica sp. nov. is proposed.

4. Protologue

Mixta hanseatica (ˈhan.se.ati.ca. fem., adj., singular, nominative. Pertaining to the location of the species’ first isolation Hansestadt Hamburg, a member of the north-German medieval commercial alliance “Hanse”). Mixta hanseatica is a Gram-negative, rod-shaped and oxidase-negative bacillus of 2–3μm length. Colonies appear non-hemolytic, mucoid, with depressed centre, firmly attached to the agar after over-night culture at 37°C in ambient air on Columbia sheep blood agar. Mixta hanseatica is a member of the Erwiniaceae family of the order of Enterobacterales. The type strain X22927 was isolated from human blood and is deposited at German Collection of Microbes and Cell cultures (DSMZ) with the accession number DSM 115429 and the National Collection of Type Cultures of the UK Health Security Agency (NCTC) with the accession number NCTC 14857. The genome sequences of the type strain’s chromosome and its four plasmids are deposited at NCBI GenBank under the accession number CP082904 (chromosome) and CP082905-CP082908 (plasmids). The ribosomal 16S-rRNA Gene is designated the identifier K6958_02190 in the genome sequence.

5. Conclusion

Isolate X22927 displayed <95% average nucleotide identity and a digital DNA–DNA hybridization value <70% compared to the closest related species of the genus Mixta, Mixta theicola. Thus we here report to the best of our knowledge the first case of a human infection by Mixta hanseatica sp. nov..

Members of the Erwiniaceae family obviously have a low propensity for causing infections in humans despite their ubiquitous appearance in the environment, suggesting limited pathogenicity. The reported number of cases of infection by Erwiniaceae other than Pantoea spp. is very low yet misidentification may lead to an underestimation of the true frequency. To date only one case of human infection with Mixta sp., a child with a pleural empyema, has been published [11]. Another genus within the Erwiniaceae is Tatumella which has been implicated in a total of four cases of septicemia in Brazil, Nigeria and Malaysia [[12], [13], [14]]. Erwinia persicina has been found as a pathogen in biliary infection [15] and urinary tract infection [16], while Erwinia billingiae was the causative agent of septic arthritis after penetrating trauma [17] and in a case of cutaneous infection with bacteremia [18]. A not fully classified isolate of Erwinia sp. has been associated with lymphadenitis [19]. Factors predisposing patients to infections with these low virulent bacteria will have to be determined in the future. As with our patient, the outcome of infections by this group of pathogens appears to be favorable [17].

Nucleotide sequence accession number

The genome sequences of the chromosome and the four plasmids were deposited in Genbank under accession numbers CP082904 (chromosome) and CP082905-CP082908 (plasmids).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Patricia Schlagenhauf

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Multimedia component 2

Pair-wise comparisons of the Mixta hanseatica sp. nov. strain X22927 and all strains which were included in the phylogenetic analysis. dDDH values according to formula d0, d4 and d6, as well as their respective difference in G+C content are given.