Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy.

Background: Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Methods: Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. Results: We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. Conclusions: We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Culturomics reveals a hidden world of vaginal microbiota with the isolation of 206 bacteria from a single vaginal sample.

The composition of the vaginal microbiota is known to be influenced by various factors and to be associated with several disorders affecting women's health. Although metagenomics is currently a widely used method for studying the human microbiota, it has certain limitations, such as a lack of information on bacterial viability. It is therefore important to use culture-based methods such as culturomics. Here, we used 35 different culture conditions to comprehensively characterize the vaginal bacterial diversity of a single woman's flora. A total of 206 bacterial species, belonging to six phyla (for a little more than half to Firmicutes, followed mainly by Actinobacteria, Bacteroidetes, and Proteobacteria) and 45 families, and 2 fungal species were cultivated. While several species of lactobacilli have been isolated, a wide variety of other bacteria were also separated, including 65 never reported before in vaginal flora, including a new bacterial species, Porphyromonas vaginalis sp. nov. Extensive culture-based methods are essential to establish a comprehensive, evidence-based repertoire of bacterial viability. If combined with molecular methods, they can provide a much more thorough understanding of the vaginal microbiota and fulfil the unknown part of metagenomic studies.

Relationship between Bacterial Vaginosis and Sexually Transmitted Infections: Coincidence, Consequence or Co-Transmission?

Sexually transmitted infections (STIs) are a serious global problem, causing disease, suffering, and death. Although bacterial vaginosis (BV) is not considered to be an STI, it may be associated with an increased risk of contracting a wide range of STIs. We sought to assess the link between the different microorganisms involved in STIs and BV. A total of 290 vaginal swabs from 290 women sent for diagnostic purposes to the clinical microbiology laboratory of the Marseille University Public Hospitals were tested by specific qPCR targeting STI-causing microorganisms and BV. Of these 290 swabs, 15.2% (44/290) were diagnosed with at least one STI-causing microorganism and 17.2% (50/290) with BV. The prevalence of STIs was significantly higher in women with BV (28%, 14/50) than in those without (20.4%, 51/240). The prevalence of co-infections involving two STI-causing microorganisms was significantly more frequent in women with BV than in those without (18% [8/50] vs. 2% [5/250]; p < 0.001). The prevalence of monoinfections and polyinfections with STI-causing microorganisms was lower in women without BV than in those with (8.8% [21/240] vs. 28% [14/50]), p < 0.001 and 2% (5/240) vs. 8% (4/50), p = 0.05, respectively). Our data suggest that a correlation between BV and STI may exist, with a higher prevalence of both monoinfections and polyinfections involving STI-causing microorganisms in women with BV. Further research is needed to better understand BV and its links to STIs.

Draft genomes and descriptions of Urmitella timonensis gen. nov., sp. nov. and Marasmitruncus massiliensis gen. nov., sp. nov., isolated from severely malnourished African children using culturomics.

Two strains, designated as Marseille-P2918T and Marseille-P3646T, were isolated from a 14-week-old Senegalese girl using culturomics: Urmitella timonensis strain Marseille-P2918T (= CSUR P2918, = DSM 103634) and Marasmitruncus massiliensis strain Marseille-P3646T (= CSUR P3646, = CCUG72353). Both strains were rod-shaped, anaerobic, spore forming motile bacteria. The 16S rRNA gene sequences of strains Marseille-P2918T (LT598554) and Marseille-P3646T (LT725660) shared 93.25% and 94.34% identity with Tissierella praeacuta ATCC 25539T and Anaerotruncus colihominis CIP 107754T, their respective phylogenetically closest species with standing in nomenclature. Therefore, strain Marseille-P2918T is classified within the family Tissierellaceae and order Tissierellales whereas strain Marseille-P3646T is classified within the family Oscillospiraceae and order Eubacteriales. The genome of strain Marseille-P2918T had a size of 2.13 Mb with a GC content of 50.52% and includes six scaffolds and six contigs, and that of strain Marseille-P3646T was 3.76 Mbp long consisting of five contigs with a 50.04% GC content. The genomes of both strains presented a high percentage of genes encoding enzymes involved in genetic information and processing, suggesting a high growth rate and adaptability. These new taxa are extensively described and characterised in this paper, using the concept of taxono-genomic description.

Antimicrobial susceptibility testing for Gram positive cocci towards vancomycin using scanning electron microscopy.

The rapid detection of resistant bacteria has become a challenge for microbiologists worldwide. Numerous pathogens that cause nosocomial infections are still being treated empirically and have developed resistance mechanisms against key antibiotics. Thus, one of the challenges for researchers has been to develop rapid antimicrobial susceptibility testing (AST) to detect resistant isolates, ensuring better antibiotic stewardship. In this study, we established a proof-of-concept for a new strategy of phenotypic AST on Gram-positive cocci towards vancomycin using scanning electron microscopy (SEM). Our study evaluated the profiling of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus after brief incubation with vancomycin. Sixteen isolates were analysed aiming to detect ultrastructural modifications at set timepoints, comparing bacteria with and without vancomycin. After optimising slides preparation and micrographs acquisition, two analytical strategies were used. The high magnification micrographs served to analyse the division of cocci based on the ratio of septa, along with the bacterial size. Susceptible strains with vancomycin showed a reduced septa percentage and the average surface area was consequently double that of the controls. The resistant bacteria revealed multiple septa occurring at advanced timepoints. Low magnification micrographs made it possible to quantify the pixels at different timepoints, confirming the profiling of cocci towards vancomycin. This new phenotypic AST strategy proved to be a promising tool to discriminate between resistant and susceptible cocci within an hour of contact with vancomycin. The analysis strategies applied here would potentially allow the creation of artificial intelligence algorithms for septa detection and bacterial quantification, subsequently creating a rapid automated SEM-AST assay.

Description of Ornithinibacillus massiliensis sp. nov., Isolated from a Child with Marasmus.

A Gram-positive, aerobic, motile, endospore-forming, rod-shaped bacterium was isolated from a stool sample of a child with marasmus. The 16S rRNA gene showed that strain Marseille-P3601T exhibited 98.68% sequence identity with Ornithinibacillus scapharcae strain TW25. The genomic DNA G+C contents of this strain was 36.9 mol%. The fatty acid profiles of the strain were iso/anteiso branched structures. The highest DDH value was 20.6%, shared with O. californiensis, amongst its closest strain phylogenetically. Based on the phylogenetic position and the genomic, morphological, and biochemical properties, strain Marseille-P3601T (=CSUR P3601=CCUG 71291) represents a novel species in the genus Ornithinibacillus, for which the name Ornithinibacillus massiliensis sp. nov. is proposed.