Proof of concept: real-time viability and metabolic profiling of probiotics with isothermal microcalorimetry.

Isothermal microcalorimetry (IMC) is a potent analytical method for the real-time assessment of microbial metabolic activity, which serves as an indicator of microbial viability. This approach is highly relevant to the fields of probiotics and Live Biotherapeutic Products (LBPs), offering insights into microbial viability and growth kinetics. One important characteristic of IMC is its ability to measure microbial metabolic activity separately from cellular enumeration. This is particularly useful in situations where continuous tracking of bacterial activity is challenging. The focus on metabolic activity significantly benefits both probiotic research and industrial microbiology applications. IMC's versatility in handling different media matrices allows for the implementation of viability assessments under conditions that mirror those found in various industrial environments or biological models. In our study, we provide a proof of concept for the application of IMC in determining viability and growth dynamics and their correlation with bacterial count in probiotic organisms. Our findings reinforce the potential of IMC as a key method for process enhancement and accurate strain characterization within the probiotic sector. This supports the broader objective of refining the systematic approach and methods used during the development process, thereby providing detailed insights into probiotics and LBPs.

Structural annotation of the conserved carbohydrate esterase vb_24B_21 from Shiga toxin-encoding bacteriophage Φ24B.

Shiga toxin-encoding bacteriophages transfer Shiga toxin genes to Escherichia coli and are responsible for the emergence of pathogenic bacterial strains that cause severe foodborne human diseases. Gene vb_24B_21 is the most highly conserved gene across sequenced Shiga bacteriophages. Protein vb_24B_21 (also termed 933Wp42 and NanS-p) is a carbohydrate esterase with homology to the E. coli chromosomally encoded NanS that deacetylates sialic acid in the intestinal mucus. To assist the functional characterization of vb_24B_21, we have studied its molecular structure by homology modelling its esterase domain and by elucidating the crystal structure of its uncharacterized C-terminal domain at the atomic resolution of 0.97 Å. Our modelling confirms that NanS from the E. coli host is the closest structurally characterized homolog to the esterase domain of vb_24B_21. Like NanS, vb_24B_21 has an atypical active site, comprising a simple catalytic dyad Ser-His and a divergent oxyanion hole. The crystal structure of the C-terminal domain reveals a lectin-like, jelly-roll ß-sandwich fold. The domain displays a prominent cleft that bioinformatics analysis predicts to be a carbohydrate binding site without catalytic properties. In summary, our study indicates that vb_24B_21 is a NanS-like atypical esterase that is assisted by a carbohydrate-binding module of yet undetermined binding specificity.

Rapid Phenotypic Antibiotic Susceptibility Testing of Uropathogens Using Optical Signal Analysis on the Nanowell Slide.

Achieving fast antimicrobial susceptibility results is a primary goal in the fight against antimicrobial resistance. Standard antibiotic susceptibility testing (AST) takes, however, at least a day from patient sample to susceptibility profile. Here, we developed and clinically validated a rapid phenotypic AST based on a miniaturized nanotiter plate, the nanowell slide, that holds 672 wells in a 500 nl format for bacterial cultivation. The multitude of nanowells allows multiplexing with a panel of six antibiotics relevant for urinary tract infections. Inclusion of seven concentrations per antibiotic plus technical replicates enabled us to determine a precise minimum inhibitory concentration for 70 clinical uropathogenic Escherichia coli isolates. By combining optical recordings of bacterial growth with an algorithm for optical signal analysis, we calculated Tlag, the point of transition from lag to exponential phase, in each nanoculture. Algorithm-assisted analysis determined antibiotic susceptibility as early as 3 h 40 min. In comparison to standard disk diffusion assays, the nanowell AST showed a total categorical agreement of 97.9% with 2.6% major errors and 0% very major errors for all isolate-antibiotic combination tested. Taking advantage of the optical compatibility of the nanowell slide, we performed microscopy to illustrate its potential in defining susceptibility profiles based on bacterial morphotyping. The excellent clinical performance of the nanowell AST, combined with a short detection time, morphotyping, and the very low consumption of reagents clearly show the advantage of this phenotypic AST as a diagnostic tool in a clinical setting.

Transcriptomic analysis of Shiga-toxigenic bacteriophage carriage reveals a profound regulatory effect on acid resistance in Escherichia coli.

Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli.

Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B.

BACKGROUND: Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT)™ and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle. RESULTS: Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network. CONCLUSION: Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.