Sliding motility of Bacillus cereus mediates vancomycin pseudo-resistance during antimicrobial susceptibility testing

Abstract

Background

The glycopeptide vancomycin is the antimicrobial agent-of-choice for the treatment of severe non-gastrointestinal infections with members of Bacillus cereus sensu lato (s.l.). Recently, sporadic detection of vancomycin-resistant phenotypes emerged, mostly for agar diffusion testing such as the disc diffusion method or gradient test (e.g. Etest^®) method.

Results

In this work, we were able to disprove a preliminarily assumed high resistance to vancomycin in an isolate of B. cereus s.l. using broth microdilution and agar dilution. Microscopic imaging during vancomycin susceptibility testing showed spreading towards the inhibition zone, which strongly suggested sliding motility. Furthermore, transcriptomic analysis using RNA-Seq on the nanopore platform revealed several key genes of biofilm formation (e.g. calY, tasA, krsEABC) to be up-regulated in pseudo-resistant cells, substantiating that bacterial sliding is responsible for the observed mobility. Down-regulation of virulence (e.g. hblABCD, nheABC, plcR) and flagellar genes compared with swarming cells also confirmed the non-swarming phenotype of the pseudo-resistant isolate.

Conclusions

The results highlight an insufficiency of agar diffusion testing for vancomycin susceptibility in the B. cereus group, and reference methods like broth microdilution are strongly recommended. As currently no guideline mentions interfering phenotypes in antimicrobial susceptibility testing of B. cereus s.l., this knowledge is essential to obtain reliable results on vancomycin susceptibility. In addition, this is the first report of sliding motility undermining accurate antimicrobial susceptibility testing in B. cereus s.l. and may serve as a basis for future studies on bacterial motility in susceptibility testing and its potential impact on treatment efficacy.

Introduction

Bacillus cereus is an underestimated pathogen that receives only marginal attention in clinical microbiology.¹ It is a ubiquitous Gram-positive and endospore-forming bacterium within a group of closely related species called Bacillus cereus sensu lato (s.l.).²

Although B. cereus s.l. is widely regarded as a dreaded food-borne pathogen, it is also the cause of various forms of local or systemic infections in both immunocompetent and immunosuppressed patients, including cutaneous and soft tissue infections, endophthalmitis, respiratory infections, osteomyelitis, endocarditis, CNS infections and bloodstream infections.^1,3

Patients with surgical or traumatic wounds, immunosuppression, haematological malignancies, indwelling catheters, IV drug abuse and (preterm) neonates are at particular risk of developing B. cereus s.l. infections.^1,4 A nosocomial origin of infections could also be demonstrated, e.g. via contaminated surfaces.^5,6 However, the fact that it is still often stigmatized as contaminant in clinical samples does not contribute to greater awareness of its potential as a pathogen.^1,3,7

In contrast to the self-limiting nature of food poisonings, non-gastrointestinal infections with B. cereus s.l. may require rapid initiation of anti-infective therapy. As non-anthrax B. cereus s.l. strains usually possess MBLs located on their chromosome,⁸ vancomycin, a glycopeptide antibiotic that inhibits the bacterial cell wall synthesis, is the agent-of-choice for empirical therapy.⁹ To ensure the optimal therapeutic approach, reliable antimicrobial susceptibility testing, including vancomycin, is necessary. However, broad knowledge about antimicrobial susceptibility of B. cereus s.l. is still limited. Although CLSI M45 guidelines have included MIC breakpoints for Bacillus spp. since 2007,¹⁰ standardized breakpoints and zone diameters from EUCAST were only introduced in 2021.¹¹ Consequently, the susceptibility testing itself of B. cereus s.l. is still poorly studied. Therefore, the need for studies on antimicrobial susceptibility in B. cereus s.l. is evident to support the clinical diagnostics.

By screening a multitude of clinical and environmental B. cereus s.l. isolates, we identified a prima facie vancomycin-resistant B. cereus s.l. strain. Both the disc diffusion method and Etest^® resulted in a phenotype highly resistant to vancomycin. To date, vancomycin resistance in B. cereus s.l. has hardly been investigated, although some studies reported resistant isolates.^8,12,13

In this work, we disproved the putative resistance and discovered that bacterial motility was responsible for mimicking vancomycin resistance. Initially, we suspected a swarming motility similar to other disc diffusion-undermining bacteria. Instead, we were able to show that sliding motility was responsible for pseudo-resistance. By using RNA-Seq on the nanopore sequencing platform, we identified a gene expression profile clearly associated with biofilm formation and bacterial sliding. Although surface mobility (e.g. swarming) has been shown to contribute to increased antibiotic tolerance in various bacteria,¹⁴ this is to our knowledge the first description of pseudo-resistance to vancomycin due to sliding motility in B. cereus s.l. and provides important insights into mechanisms that interfere with antimicrobial susceptibility testing in the B. cereus group.

Materials and methods

Bacterial strains and growth conditions

The B. cereus group isolate BC70 was isolated from food packaging material as described previously.¹⁵ The mesophilic strain belonged to phylogenetic group IV, defining the isolate as Bacillus cereus sensu stricto (s.s.) according to the current nomenclature.² The type strain ATCC 14579 (DSM31) was used as negative control (Leibniz Institute DSMZ, Germany).

Antimicrobial susceptibility testing

The disc diffusion method was performed in accordance with current EUCAST guidelines¹⁶ with the following antimicrobial agents (BBL^TM Sensi-Disc^TM, Becton Dickinson): vancomycin (5 µg), clindamycin (2 µg), erythromycin (15 µg), linezolid (10 µg), imipenem (10 µg), meropenem (10 µg), ciprofloxacin (5 µg) and levofloxacin (5 µg). To determine the MIC of vancomycin and teicoplanin, gradient tests (Etest^® Vancomycin, bioMérieux; MIC Test strip Teicoplanin, Liofilchem) were used. Broth microdilution using the Bioscreen C system (Oy Growth Curves Ab Ltd) and agar dilution were performed as reference methods, both based on EUCAST recommendations.^17,18 The Mueller–Hinton (MH, Carl Roth) broth and the MH agar dilution plates were supplemented with vancomycin hydrochloride (Heraeus Medical) to reach the following final concentrations: 0.125, 0.25, 0.5, 1, 2, 4, 8 and 16 mg/L. For broth microdilution and agar dilution assays, all concentrations were tested in five replicates and growth controls were included.

Time-lapse microscopy

The growth behaviour of BC70 during agar diffusion susceptibility testing was visualized by time-lapse microscopy overnight using the Nikon High Content Screening System (Nikon Eclipse Ti2 microscope) with microscope cage incubator (Okolab). Vancomycin gradient tests were applied on MH agar plates and were incubated for 24 h at 37°C. Images of the region of interest were automatically taken every 30 min with Plan Apo 4× objective lenses. Images were taken with Zyla sCMOS camera (Andor) and image processing was carried out with NIS elements viewer and Fiji (v2.15.0).

Phase-contrast microscopy

The morphology of inhibition zone edges was further investigated with phase-contrast microscopy. For this purpose, approximately 1.5 × 1.5 cm sections of the vancomycin gradient test plates containing the region of interest were prepared with a scalpel, placed on a microscope slide and covered with a cover slip. Microscopy was carried out on the Axio Lab.A1 microscope (Zeiss) using 40× phase-contrast objective lenses.

Testing for swarming motility

Leifson flagella stain including counterstaining with Loeffler methylene blue solution (Merck) was used to visualize the flagella in swarming cultures.¹⁹ Bright-field microscopy was carried out on the Axio Lab.A1 microscope using 100× objective lenses and oil immersion. Swarming cells of the BC70 isolate and ATCC 14579 were used as positive controls. Bacteria were spotted on tryptone swarming agar (TrA) containing 1% (w/v) tryptone (Oxoid), 0.5% (w/v) NaCl and 0.7% (w/v) agar, and incubated at 37°C in a humidified chamber until swarming motility was observed. Swarming bacteria were then subcultured on TrA and Mueller–Hinton swarming agar (MHS, 0.7% agar, w/v) to obtain stable swarming populations.

Colony expansion assay

The colony expansion was assessed by spotting 5 µL of a bacterial suspension equivalent to McFarland 0.5 on Columbia Agar with 5% Sheep Blood (COL-S, Becton Dickinson) agar, MH agar and TrA medium. All plates were incubated at 37°C and evaluated every 24 h. The TrA plates were incubated in a humidified chamber. All measurements were done in triplicates.

RNA-Seq

The BC70 isolate and ATCC 14579 were cultured on MH agar with vancomycin gradient tests. Furthermore, a swarming phenotype of BC70 on MHS agar and cultures on normal MH plates were prepared. All samples were taken after exactly 13 h of incubation, as motility on vancomycin gradient test plates towards the inhibition zone could be observed by time-lapse microscopy at this time. For each condition, two to four biological replicates were pooled. For vancomycin gradient test plates, cells were taken from both sides of the Etest^® strip at the innermost inhibition zone edges (Figure S1, available as Supplementary data at JAC Online). The swarming cells were confirmed by staining the flagella and picked from the outermost edges of swarming colonies. About five to six colonies were taken from each normal MH plate. The protocols for RNA isolation, library preparation and nanopore sequencing are described in more detail in the Supplemental Material and methods. Differential expression analysis was performed with EPI2ME Desktop (v5.0.2). Data from the respective conditions were compared with each other, e.g. BC70 isolate against ATCC 14579. Functional analysis for gene ontology (GO) was performed using FUNAGE-Pro.²⁰ Furthermore, genes were manually classified according to their proposed function using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, analogous genes in other B. cereus group strains and profound literature research. Refer to Supplemental Material and methods for a detailed description of RNA-Seq data analysis. Gene expression data were deposited in the NCBI Gene Expression Omnibus repository (accession no. GSE253142).

Results

Vancomycin pseudo-resistant phenotype

The putatively vancomycin-resistant isolate BC70 was identified while screening numerous food packaging isolates for their antimicrobial susceptibility. The disc diffusion test included several clinically relevant antibiotic substances. The vancomycin disc (5 µg) showed no zone of inhibition (Figure 1a), whereas all other antibiotics were reported as susceptible. The subsequent vancomycin gradient tests revealed a MIC of 24 to 64 mg/L (Figure 1b), suggesting a highly resistant phenotype. A parallel MIC determination of the glycopeptide teicoplanin using test strips resulted in a MIC of 4 mg/L (Figure S2). We therefore performed a broth microdilution for vancomycin, the gold standard for MIC determination, which resulted in a MIC of 4 mg/L (Figure 1c). Subsequent agar dilution confirmed a MIC of 4 mg/L (Figure 1d). As a result, the putative resistance was disproved and was not related to solid media, but a consequence of agar diffusion susceptibility testing using disc diffusion or the gradient test method. In parallel, susceptibility tests were performed on the B. cereus reference strain ATCC 14579, which yielded a susceptible result in the disc diffusion test and vancomycin MICs of 2 mg/L in the gradient test, broth microdilution and agar dilution.

Figure 1.

B. cereus isolate BC70 displayed pronounced discrepancy in vancomycin susceptibility using different methods. (a) Disc diffusion method, arrow indicates vancomycin disc. (b) Gradient test method with a vancomycin test strip, MIC = 64 mg/L. (c) Broth microdilution, MIC = 4 mg/L. (d) Agar dilution, MIC = 4 mg/L. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Microscopy revealed bacterial movement but not swarming

Growth of BC70 exposed to a vancomycin gradient test was monitored for 24 h using time-lapse microscopy. We observed growth up to a MIC of 2 mg/L vancomycin after 6 h (Figure 2), which, however, continuously expanded in the following hours. After 8 h, the EUCAST breakpoint of 2 mg/L was exceeded, and reached a MIC of 24 mg/L after 16 h. The BC70 isolate could visibly move into the initial inhibition zone covering a distance of more than 0.5 mm within 1 h (Supplemental Movie). The rapidly spreading phenotype was also observed when comparing the size of colonies over time on MH agar and blood agar (Figure 3).

Figure 2.

Time-lapse microscopic growth monitoring revealed continuous expansion of BC70 cells on MH agar with vancomycin gradient test. The respective MIC is labelled, starting at 2 mg/L after 6 h and ending at 32 mg/L after 22 h of incubation. The highest point with visible growth along the strip was used to determine the MIC.

Figure 3.

The BC70 isolate showed a strong ability to spread on the agar surfaces of different growth media compared with reference strain ATCC 14579. The following media were used: Columbia agar with 5% sheep blood (COL-S), Mueller–Hinton agar (MH) and tryptone swarming agar (TrA). Colony diameters (mm) are shown with boxplots including individual data points. Maximal plate diameter was 85 mm. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

The cells that were moving into the inhibition zone showed a more translucent growth compared with cells growing at the periphery of the gradient test. Light microscopy revealed that expanding colonies formed a thin layer of advancing cells rather than dense colonies (Figure 4a). A detailed examination of the colony rims by light microscopy and phase-contrast microscopy revealed a migration front composed of so-called van Gogh bundles, finger-like extensions or waves of elongated cells that are tightly aligned (Figure 4a and 4b).²¹ Specific flagellar staining demonstrated a complete absence of flagella in cells at the migration front (Figure 4c), and the cells that initially appeared to be solely elongated turned out to be multiple cells arranged in long chains with a clearly visible septation. In contrast, swarming cells from MHS medium were clearly hyperflagellated and elongated (Figure 4d). Altogether, microscopic imaging revealed a spreading mechanism of BC70 that is different from swarming and strongly suggests sliding motility due to the presence of van Gogh bundles.

Figure 4.

(a) BC70 formed thin layers of cells moving towards the gradient test. (b) Phase-contrast microscopy of BC70 on MH with a vancomycin gradient test showed wave-like colony rim and elongated cell bundles. (c) Flagella stain of BC70 on MH with a vancomycin gradient test disproved swarming motility and revealed long chains of B. cereus cells. (d) Distinct swarming phenotype with hyperflagellated cells of BC70 on swarming medium (MHS). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Transcriptomic analysis identified biofilm gene expression during susceptibility testing

To learn more about the observed mobility and simultaneously confirm the non-swarming phenotype, RNA-Seq transcriptomic analysis for differential gene expression was performed via nanopore sequencing. The BC70 and ATCC 14579 cells were collected from the edges of the respective inhibition zone after exactly 13 h of incubation on plates with a vancomycin gradient test to study actively growing cells during antimicrobial susceptibility testing. The RNA-Seq analysis identified that 164 genes were differentially expressed (fold change at least  ± 2) at a statistically significant level [false discovery rate (FDR) adjusted P value <0.05]. More specifically, 104 genes in BC70 cells were up-regulated whereas 60 were down-regulated compared with ATCC 14579 cells. Among the differentially expressed genes, a functional classification based on GO revealed 25 significantly overrepresented GO terms (Figure 5a). Pathogenesis, haemolysis and toxin activity were shown to be down-regulated, whereas sporulation was up-regulated. Moreover, genes assigned to the GO terms of antibiotic biosynthetic process and glycogen biosynthetic process showed increased expression, as did genes for DNA transposition processes. The large number of genes assigned to the extracellular region indicated relevant changes in transportation and secretion processes involved in mimicking a vancomycin resistance in agar diffusion tests. However, only 63 genes could be assigned to a GO classification.

Figure 5.

Transcriptomic analysis revealed biofilm-associated gene expression in motile BC70 cells during vancomycin agar diffusion testing. (a) Overrepresented GO terms in BC70 cells with exposure to a vancomycin gradient test relative to reference strain ATCC 14579 with vancomycin gradient test. (b) Differentially expressed genes between BC70 and ATCC 14579 clustered according to their biological functions as proposed in the literature. Genes could be assigned to one or more functional groups. (c) Log₂ fold change in gene expression of selected biofilm and virulence genes in BC70 relative to ATCC 14579. (d) Heat map of differentially expressed genes (log₂ fold change) of ATCC 14579 with exposure to a vancomycin gradient test, BC70 swarming cells and BC70 on plain MH agar compared with BC70 (with vancomycin gradient test). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Manual classification of the up-regulated genes revealed genes directly involved in biofilm formation or biofilm-associated genes, as well as putative quorum-sensing genes and several sporulation genes (Figure 5b). The genes for the biofilm matrix proteins calY and tasA had more than 4- and 21-fold higher transcription, respectively, than in ATCC 14579 cells (Figure 5c). The calY gene also showed the highest statistical significance of all differentially expressed genes here (FDR adjusted P value = 7.56 × 10⁻⁴¹). Further up-regulated genes associated with biofilm formation were the krs operon encoding the lipopeptide kurstakin [increased expression ranging from 25-fold (krsC) up to 281-fold (krsA)]. Additionally, the biofilm-associated regulators encoded by mogR and comER showed significantly increased expression. There were also 12 up-regulated genes encoding proteins involved in sporulation and its regulation including spo0M, spoIIAA, spoIIAB, spoIIE, spoIVA, spoVG and sigF. Besides sporulation, the expression of spoVG is also crucial for biofilm formation.²² Based on literature and database search, three up-regulated genes could be linked to putative quorum-sensing systems and one gene coding for a flagellin was found.

Conversely, the 60 down-regulated genes included genes related to multiple virulence factors as well as genes previously associated with planktonic cells or down-regulated in B. cereus biofilms (Figure 5b). Toxin-related genes were particularly prominent among the down-regulated genes including the nhe operon, the hbl operon and the cereolysin-encoding gene clo (Figure 5c). Other significantly down-regulated virulence genes were immune inhibitor inhA2 and the neutral proteases nprP2 and nprB. The metabolism-related genes pruA, rocD, hutI and gcvPA, the gene lldp coding for L-lactate permease, and the transporter-encoding gene gltP as well as some genes encoding proteins of the tricarboxylic acid cycle were down-regulated. These genes were previously shown to be associated with planktonic cells or were expressed at decreased levels in B. cereus biofilms.²³ Gene expression for ribosome and protein biosynthesis hardly differed between BC70 and ATCC 14579. A full list of differentially expressed genes is provided in the Supplemental material (List_of_differentially_expressed_genes.xlsx).

The gene expression profile of BC70 cells during vancomycin agar diffusion testing was thus substantially different from the expression profile of ATCC 14579. To verify the microscopy finding that BC70 did not display a swarming phenotype upon agar diffusion tests, we compared its gene expression profile with the transcriptome of swarming BC70. A total of 377 differentially expressed genes were successfully identified. Among 191 up-regulated genes in swarming cells, we discovered 23 genes directly involved in the flagellar system (Figure 5d). Besides, numerous virulence genes were widely up-regulated, whereas sporulation genes and biofilm-affecting genes such as calY, sipW and mogR were substantially down-regulated in swarming cells. Notably, sinR, which codes for a biofilm repressor, was also down-regulated in swarming cells. Additionally, the normal expression profile of BC70 cells on MH agar without any vancomycin was compared with BC70 from the gradient test plate. Again, we found a significant up-regulation of virulence genes and down-regulation of biofilm- and sporulation-associated genes at the same time. Both the swarming cells and the MH cells showed enhanced expression of genes related to ribosomes and protein biosynthesis, indicating a more active metabolism compared with the cells at the edge of the inhibition zone. The RNA-Seq results confirmed that BC70 uses a mechanism distinct from swarming motility to spread in the inhibition zone and revealed a biofilm-associated gene expression pattern providing evidence to support the sliding motility.

Discussion

Numerous publications have reported a 100% susceptibility of B. cereus s.l. isolates to vancomycin,^6,24–27 whereas true vancomycin resistance is rather a sporadic exception. Notably, vancomycin-resistant isolates were often detected when the disc diffusion method was applied.^12,13,28 Due to the fast and straightforward application of disc diffusion, this method is broadly used for susceptibility testing, and the release of standardized zone diameters by EUCAST v11.0 in 2021 may further contribute to the propagation of disc diffusion as it is no longer necessary to fall back on Staphylococcus aureus zone diameters from CLSI M100 guidelines.^11,29 A major drawback is that putative resistance in disc diffusion is often confirmed by gradient testing. Reference methods like broth microdilution are rarely used in routine microbiology laboratory settings due to their required labour input and costs. However, based on our findings, this seems to be necessary before reporting a strain of B. cereus s.l. to be resistant to vancomycin.

Mills et al.⁸ recently reported two vancomycin-resistant isolates using broth microdilution and at the same time demonstrated the insufficiency of disc diffusion tests for the B. cereus group. In addition, the mere detection of genes suspected to be associated with vancomycin resistance has never provided evidence of phenotypic resistance in B. cereus.^8,25,30 We therefore strongly recommend phenotypical methods independent from agar diffusion to confirm a putative vancomycin resistance. This is of utmost importance, as vancomycin is considered the drug-of-choice in the treatment of serious infections with B. cereus group species.^4,9,31

This study also aimed to understand the mechanisms responsible for the misinterpretation of vancomycin agar diffusion testing in the B. cereus group. Although a fixed concentration of vancomycin above the MIC consistently prevented growth, methods that rely on a vancomycin gradient in solid media such as disc diffusion or gradient tests may allow expansion of B. cereus s.l. on the agar surface. Swarming motility is a well-known strategy of various bacteria to actively move on surfaces and should be cautiously handled when interpreting inhibition zones.¹⁶ Although its ability for swarming motility has been shown,³²  B. cereus s.l. has not yet been recognized as a swarming species that could interfere with the agar diffusion test. Instead of swarming motility, however, we discovered that isolate BC70 was able to spread over the agar surface into the inhibition zone via sliding motility. Bacterial sliding is a passive mechanism that is mainly driven by the expansive forces of cell division and the synthesis of friction-reducing components, e.g. surfactants.³³

During surface translocation of BC70, we found a gene expression pattern typically associated with biofilm cells.²³ The GO analysis revealed pronounced differences in the extracellular region of BC70 compared with ATCC 14579, which was reflected in the increased expression of calY and tasA, encoding two biofilm matrix proteins that form structural fibrils in the extracellular matrix.^34,35 Their homologue in B. subtilis, tasA, is essential for proper colony expansion and is likely to be involved in fine-tuning the formation of van Gogh bundles during bacterial sliding.²¹ The lipopeptide kurstakin, encoded by the krs locus, is involved in proper biofilm structuration.³⁶ In general, lipopeptides are known for their surfactant properties and therefore play a major role in reducing surface tension for bacterial motility and biofilm formation.³⁷ Similar to surfactin in B. subtilis, kurstakin may function as the surfactant that is essential for sliding motility.^21,38,39 As in other studies on B. cereus biofilms, however, not all transcripts of actually co-expressed genes could be detected, which demonstrates how variable gene expression studies on biofilms and motility still are.^23,40,41 Enhanced transcription of eps operons, which are related to the production of extracellular polysaccharides and necessary for sliding motility and van Gogh bundles, was not detected.^21,38

Increased antimicrobial resistance is a common characteristic of biofilms and has also been demonstrated for swarming cells due to high cell densities paired with mobility.¹⁴ This may act synergistically with a putative barrier function of a surfactant to facilitate the survival of BC70 upon vancomycin exposure. Because pseudo-resistance was only observed with vancomycin, the question of whether the specific effect of vancomycin on peptidoglycan synthesis plays a role or whether the cells are shielded from vancomycin by biofilm matrix or surfactant remains unanswered. The fact that biofilm and sporulation pathways are actually intertwined is well reflected by the increased expression of the regulator-encoding genes spoVG and comER.^22,42 SpoVG has been shown to regulate the transcription levels of Spo0A, which in turn governs sliding motility in B. subtilis.³⁸ In fact, Grau et al.³⁸ showed a sequential transition from sliding motility to sessile biofilm and sporulation, which explains the up-regulation of genes related to sporulation in this study. Sliding motility accompanied by van Gogh bundles has been described in B. cereus under nutrient starvation.³⁹ Consequently, stress such as antibiotic exposure could facilitate a sliding phenotype. The sliding motility of B. subtilis was indeed activated upon exposure to subinhibitory concentrations of several antibiotics, but vancomycin was not tested.⁴³ In addition, sliding motility was also suspected in small colony variants of B. cereus induced upon exposure to aminoglycosides.⁴⁴

Swarming motility was eventually refuted not only due to the absence of hyperflagellated cells, but also due to increased expression of mogR, coding for a transcriptional repressor of flagellar motility and virulence that promotes biofilm gene expression.⁴⁵ Furthermore, swarming motility has been shown to be associated with increased expression of PlcR-regulated virulence genes, which was confirmed in our study.⁴⁶ This is supported by the fact that PlcR represses surfactant production and sliding motility.³⁹

A very similar phenotype in Bacillus thuringiensis has recently been termed the ‘fuzzy spreader’ morphotype, which has been described as biofilm-specialist with enhanced biofilm productivity and translocation.⁴⁷ However, whether this morphotype also exhibits sliding motility and how it is affected by antibiotic exposure remains to be investigated.

Because sliding motility appears to be an effective strategy of the B. cereus group to evade in vitro exposure to vancomycin, in vivo experiments on possible effects of sliding motility in B. cereus infections may contribute to understanding its role in a clinical context. Finally, it should be noted that the mechanism for mimicking vancomycin resistance was only investigated in the BC70 isolate. However, Baghbadorani et al.⁴⁸ reported an alarming number of 20% vancomycin-resistant isolates in disc diffusion testing, which could indicate that it is more prevalent than previously noted. Future research should therefore target the actual prevalence of false-resistant B. cereus s.l. strains in both clinical settings and the environment. This will help to ensure reliable susceptibility testing and maintain important therapeutic options for the treatment of severe B. cereus infections.

Funding

This project was funded by the Medical University of Graz and did not receive any external funding.

Transparency declarations

None to declare.

Author contributions

P.J.S. and C.K. conceived the study. P.J.S. performed all the experiments including nanopore sequencing and bioinformatic analysis. P.J.S. and P.F. wrote the manuscript. C.K. and P.F. revised and edited the manuscript. C.K. supervised the project.

Supplementary data

Supplemental Figures S1–S3, Supplemental material, Supplemental movie, and Supplemental Material and methods are available as Supplementary data at JAC Online.