Genomic insights into Leminorella grimontii and its chromosomal class A GRI β-lactamase

Leminorella grimontii strain LG-KP-E1-2-T0 was isolated from Zophobas morio larvae. It showed a susceptibility phenotype compatible with the expression of an inducible extended-spectrum β-lactamase. The presence of a chromosomal bla gene encoding for the class A GRI-1 β-lactamase was revealed by whole-genome sequencing. GRI-1 shared the highest amino acid identity with RIC-1 and OXY-type β-lactamases (76–80%). Analysis of six further publicly-available L. grimontii draft genomes deposited in NCBI revealed that blaGRI−1 was always present. Core-genome analysis indicated that LG-KP-E1-2-T0 was unique from other strains. We provided the first complete genome of L. grimontii and new insights on its chromosomal β-lactamases. Supplementary Information The online version contains supplementary material available at 10.1007/s10096-024-04888-7.


Introduction
Leminorella spp.are Gram-negative bacteria belonging to the order of Enterobacterales and the family Budviciaceae [1].So far, the Leminorella genus includes three taxa: L. grimontii, L. richardii and Leminorella sp.strain 3 [2].Among them, L. grimontii is the most frequently reported in humans [3,4].This species has been isolated in stool samples and identified as responsible for spontaneous peritonitis and neonatal sepsis [2][3][4].However, no complete genomes of L. grimontii are currently available in the NCBI database.

Analysis of GRI amino acid sequences
All bla coding sequences (CDS) confirmed by BLASTn as bla GRI were extracted from the six L. grimontii draft genomes.Subsequently, they were translated into amino acid sequences using Geneious Prime (Biomatters) v2023.2.1 (parameters: genetic code, bacterial, transl_table 11).Furthermore, the three previously deposited proteins A4FRA6, WP_027275480.1 and WP_261832807.1 and the one found in LG-KP-E1-2-T0 were used to generate a multiple sequence alignment with MUSCLE v5.1 in Geneious Prime.

Phenotypic testing
The MALDI-TOF MS identified LG-KP-E1-2-T0 as L. grimontii (score of 2.36).The strain was resistant to cefotaxime and aztreonam, but susceptible to cefoxitin, ceftriaxone, cefepime, carbapenems and βL/βLIC (Table 1).This phenotype was consistent with the production of an ESBL, as further confirmed by the results of the CDT and DDST assays (Figure S1-A/B) [10,11].Moreover, an inducible phenotype was suspected with the DDST and well-confirmed with the cefoxitin and imipenem assays (Figure S1-C/D).

The GRI-I β-lactamase
The bla GRI found in LG-KP-E1-2-T0 encoded a protein of 295 amino acid residues with all classic motifs of class A/subclass A1 β-lactamases (Fig. 2A) [8].This enzyme shared 100% similarity with those from JCM 5900 and WP_261832807.1.In contrast, compared to that of the remaining sequences, the GRI protein of LG-KP-E1-2-T0 differed by two amino acid substitutions (V17A and S23C) in the signal peptide.Since no amino acid substitutions were identified in the leader sequence, we classified all encoded GRI β-lactamases of the L. grimontii genomes as GRI-1 [30].

Conclusions
We provided the first complete genome of L. grimontii, an emerging pathogen in the clinical context [3,5].The strain was unexpectedly isolated from Z. morio larvae [9], though L. grimontii was also found in the gut microflora of mosquito and red palm weevil [31,32].
Our genome comparative analysis, along with the phenotypic confirmatory testing, suggested that all L. grimontii express an inducible chromosomally-encoded class A ESBL (GRI-1) with cefotaximase activity.Future studies should be directed at finding possible GRI variants and characterizing the kinetic properties of these enzymes.Moreover, the available complete genome of L. grimontii may be useful for larger epidemiological analyses.

Fig. 2 (
Fig. 2 (A) Structure-based protein alignment of GRI-1 β-lactamase from L. grimontii.The signal peptide and mature protein regions are delineated in black, above the sequence.At substitution site, identical amino acid residues to each other are illustrated in black.Strictly conserved motifs in class A enzymes [SXXK (active site: position 70-73), SDN (position 130-132), E and KTG (positions 166 and 234-236)], subclass A1 [RXEXXLN (position 164-170), VGDKTG (position 231-236)] are shown in light blue and framed in black, respectively.Corresponding GenBank accession numbers are given in parentheses.(B) The phylogenetic tree represents the similarity at the amino acid sequence level of 40 representative class A β-lactamases.

Table 1
Antimicrobial susceptibility profile of the L. grimontii strain