Evaluation of Automated Disk Diffusion Antimicrobial Susceptibility Testing Using Radian® In-Line Carousel.

Antimicrobial susceptibility testing (AST) by disk diffusion provides an accurate image of bacterial growth, enabling the detection of culture purity, heterogeneous growth, and antibiotic interactions. However, this manual method is time-consuming and visual interpretation is prone to errors. To overcome these disadvantages, the Radian® In-Line Carousel (Copan, Brescia, Italy) was launched, which is a WASPLab® module dedicated to full automation of (pre)-analytical steps as well as interpretation of disk diffusion AST. However, until now, no evaluation of Radian® against manual disk diffusion has been performed. We assessed the categorical agreement (CA) between standardized disk diffusion (reference method) and Radian® using EUCAST 2021 breakpoints. We tested 135 non-duplicate strains, selected from the National EUCAST challenge panel, clinical strains, and external quality controls. The strains included Enterobacterales (n = 63), Enterococcus faecalis (n = 3), Enterococcus faecium (n = 10), Pseudomonas aeruginosa (n = 16), Staphylococcus aureus (n = 19), coagulase-negative staphylococci (n = 4), and Streptococcus spp. (n = 20). Furthermore, we explored antibiotic disk thermolability in the WASP Radian® carousel by testing 10 ATCC® strains up to 7 days. The observed CA was 95.3%, 96.3%, 93.8%, 97.3% and 98.0% for Enterobacterales, Enterococcus spp., P. aeruginosa, Staphylococcus spp. and Streptococcus spp., respectively, resulting in an acceptable overall CA for all groups. (Very) major error rates were ≤ 5% for all antibiotics. Antibiotic disk thermostability was confirmed up to 4 days in the WASP Radian® In-Line Carousel. The Radian® In-Line Carousel provides a fully automated solution for accurate disk diffusion AST, reducing workload and improving standardization and traceability. In addition, our study demonstrated the thermostability of antibiotic disks up to 4 days in the WASP Radian® In-Line Carousel.

Comparing Vaginal and Endometrial Microbiota Using Culturomics: Proof of Concept.

It is generally accepted that microorganisms can colonize a non-pathological endometrium. However, in a clinical setting, endometrial samples are always collected by passing through the vaginal-cervical route. As such, the vaginal and cervical microbiomes can easily cross-contaminate endometrial samples, resulting in a biased representation of the endometrial microbiome. This makes it difficult to demonstrate that the endometrial microbiome is not merely a reflection of contamination originating from sampling. Therefore, we investigated to what extent the endometrial microbiome corresponds to that of the vagina, applying culturomics on paired vaginal and endometrial samples. Culturomics could give novel insights into the microbiome of the female genital tract, as it overcomes sequencing-related bias. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy were included. An additional vaginal swab was taken from each participant right before hysteroscopy. Both endometrial biopsies and vaginal swabs were analyzed using our previously described WASPLab-assisted culturomics protocol. In total, 101 bacterial and two fungal species were identified among these 10 patients. Fifty-six species were found in endometrial biopsies and 90 were found in vaginal swabs. On average, 28 % of species were found in both the endometrial biopsy and vaginal swab of a given patient. Of the 56 species found in the endometrial biopsies, 13 were not found in the vaginal swabs. Of the 90 species found in vaginal swabs, 47 were not found in the endometrium. Our culturomics-based approach sheds a different light on the current understanding of the endometrial microbiome. The data suggest the potential existence of a unique endometrial microbiome that is not merely a presentation of cross-contamination derived from sampling. However, we cannot exclude cross-contamination completely. In addition, we observe that the microbiome of the vagina is richer in species than that of the endometrium, which contradicts the current sequence-based literature.

Validation of the Colibrí Instrument for Automated Preparation of MALDI-TOF MS Targets for Yeast Identification.

Recently, Copan (Italy) introduced the Colibrí instrument for automated colony picking and preparation of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) target plates. Our study aimed to validate this system for yeasts as such testing has not been performed yet and is a missing link needed to implement the system for routine use. Fifty-five Candida strains were selected to evaluate the accuracy of Colibrí. For each strain, a sheep blood agar plate supplemented with X and V factors (HEM) and a Sabouraud agar plate (SAB) were inoculated and incubated using the WASPlab specimen processing system (Copan). After 18 h and 36 h of incubation, the isolates were spotted in parallel using Colibrí and manually onto MALDI-TOF target plates with the addition of formic acid and identified using MALDI-TOF mass spectrometry. The reproducibility was evaluated using ATCC reference and clinical isolate-derived strains. The cumulative percentage of acceptable identification scores (IDs) after 36 h was 91% for strains cultured on HEM plates using both Colibrí and the manual method. The SAB plates showed inferior results for both Colibrí (76%) and the manual method (78%). We observed an overall agreement of 92% at 18 h for identification of the strains on the HEM plates between Colibrí and the manual method and 94% after 36 h. For the SAB plates, the agreement was 78% after 18 h and 84% after 36 h. Apart from Candida dubliniensis and Candida tropicalis, all Candida species were identified with 100% accuracy using Colibrí on HEM plates. We observed good agreement between Colibrí and the manual reference method. These results demonstrate that Colibrí is a reliable system for MALDI-TOF target preparation for yeast identification, allowing increased standardization and less hands-on time.

Validation of Rapid Antimicrobial Susceptibility Testing directly from blood cultures using WASPLab®, including Colibrí™ and Radian® in-Line Carousel.

With the increase in antimicrobial resistance, fast reporting of antimicrobial susceptibility testing (AST) results is becoming increasingly important. EUCAST developed a method for rapid AST (RAST) directly from the broth of positive blood cultures (BC). Inhibition zones are read after 4, 6, and 8 h, with specific breakpoints per time point. We evaluated the RAST method based on EUCAST disk diffusion methodology with inoculation of BC broth using WASPLab® (inclusive Colibrí™ and Radian®). Forty-nine non-duplicate strains were tested: Escherichia coli n = 17, Klebsiella pneumoniae n = 7, Pseudomonas aeruginosa n = 4, Acinetobacter baumannii n = 2, Staphylococcus aureus n = 10, Enterococcus faecalis n = 6, and Enterococcus faecium n = 3. Results were compared to direct AST and standardized AST. Good categorical agreement was obtained at all time points for all groups, except P. aeruginosa. RAST cut-offs for extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales enabled the detection of all included ESBL isolates (n = 5) at all time points, except for 1 E. coli ESBL after 4 h. RAST cut-offs for carbapenemase-producing Enterobacterales enabled the detection of only one carbapenemase after 6 h. However, all carbapenemases (n = 3) were correctly detected after 8 h. Two methicillin-resistant S. aureus were included; both were correctly categorized as cefoxitin-resistant at 6 and 8 h. At 4 h, there was insufficient growth for inhibition zone interpretation. EUCAST RAST is a fast supplementary tool for direct AST of positive BC. WASPLab® provides a significant advantage as pictures are made automatically implicating that we are not strictly bound to the time points for inhibition zone interpretation.

Culturomics to Investigate the Endometrial Microbiome: Proof-of-Concept.

The microbiome of the reproductive tract has been associated with (sub)fertility and it has been suggested that dysbiosis reduces success rates and pregnancy outcomes. The endometrial microbiome is of particular interest given the potential impact on the embryo implantation. To date, all endometrial microbiome studies have applied a metagenomics approach. A sequencing-based technique, however, has its limitations, more specifically in adequately exploring low-biomass settings, such as intra-uterine/endometrial samples. In this proof-of-concept study, we demonstrate the applicability of culturomics, a high-throughput culturing approach, to investigate the endometrial microbiome. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy, as part of their routine work-up at Brussels IVF, were included after their informed consent. Biopsies were used to culture microbiota for up to 30 days in multiple aerobic and anaerobic conditions. Subsequent WASPLab®-assisted culturomics enabled a standardized methodology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA sequencing was applied to identify all of bacterial and fungal isolates. Eighty-three bacterial and two fungal species were identified. The detected species were in concordance with previously published metagenomics-based endometrial microbiota analyses as 77 (91%) of them belonged to previously described genera. Nevertheless, highlighting the added value of culturomics to identify most isolates at the species level, 53 (62.4%) of the identified species were described in the endometrial microbiota for the first time. This study shows the applicability and added value of WASPLab®-assisted culturomics to investigate the low biomass endometrial microbiome at a species level.

Performance of BacT/Alert resin-based FN plus bottles compared with BacT/Alert charcoal-based FN bottles for the detection of anaerobes in experimentally seeded blood cultures.

Recently new resin-based BacT/Alert FAN Plus bottles containing antibiotic-binding polymeric beads, were introduced as an improvement of the charcoal-based FAN bottles for the recovery of bacteria and fungi. To assess the performance of the novel anaerobic FN plus bottles in the detection of anaerobic organisms, we compared the detection rate and the time to detection (TTD) in spiked resin-based FN Plus bottles and charcoal-based FN bottles. The bottles were experimentally seeded with reference strains or clinical strains collected from positive blood cultures. Five reference strains and fifty-five clinically significant anaerobic isolates were investigated, of which 91% (61/67) showed growth in both types of bottles within an incubation time of 5 days. A significant prolonged median TTD of 45 h for anaerobic microorganisms was observed in the resin-based bottles versus 29 h in the charcoal-based bottles (P < 0.0001). Bacteroides spp., associated with higher virulence and higher mortality rates in bloodstream infections, were detected faster in the charcoal-based bottles as compared to the resin-based bottles. In conclusion recently improved resin FN bottles showed a significantly increased median TTD for the recovery of anaerobic isolates.