Development and validation of a quick, automated, and reproducible ATR FT-IR spectroscopy machine-learning model for Klebsiella pneumoniae typing.

The reliability of Fourier-transform infrared (FT-IR) spectroscopy for Klebsiella pneumoniae typing and outbreak control has been previously assessed, but issues remain in standardization and reproducibility. We developed and validated a reproducible FT-IR with attenuated total reflectance (ATR) workflow for the identification of K. pneumoniae lineages. We used 293 isolates representing multidrug-resistant K. pneumoniae lineages causing outbreaks worldwide (2002-2021) to train a random forest classification (RF) model based on capsular (KL)-type discrimination. This model was validated with 280 contemporaneous isolates (2021-2022), using wzi sequencing and whole-genome sequencing as references. Repeatability and reproducibility were tested in different culture media and instruments throughout time. Our RF model allowed the classification of 33 capsular (KL)-types and up to 36 clinically relevant K. pneumoniae lineages based on the discrimination of specific KL- and O-type combinations. We obtained high rates of accuracy (89%), sensitivity (88%), and specificity (92%), including from cultures obtained directly from the clinical sample, allowing to obtain typing information the same day bacteria are identified. The workflow was reproducible in different instruments throughout time (>98% correct predictions). Direct colony application, spectral acquisition, and automated KL prediction through Clover MS Data analysis software allow a short time-to-result (5 min/isolate). We demonstrated that FT-IR ATR spectroscopy provides meaningful, reproducible, and accurate information at a very early stage (as soon as bacterial identification) to support infection control and public health surveillance. The high robustness together with automated and flexible workflows for data analysis provide opportunities to consolidate real-time applications at a global level. IMPORTANCE We created and validated an automated and simple workflow for the identification of clinically relevant Klebsiella pneumoniae lineages by FT-IR spectroscopy and machine-learning, a method that can be extremely useful to provide quick and reliable typing information to support real-time decisions of outbreak management and infection control. This method and workflow is of interest to support clinical microbiology diagnostics and to aid public health surveillance.

MicroMundo@UPorto: an experimental microbiology project fostering student's antimicrobial resistance awareness and personal and social development.

Antimicrobial resistance (AMR) is a global societal challenge requiring the contribution of professionals along with general community citizens for their containment. Portugal is one of the European countries where a lack of knowledge on the correct use of antimicrobials and AMR problematic is preeminent. Moreover, youth demotivation to pursue science careers is emerging. To address these problems an innovative experimental service-learning pedagogical strategy, MicroMundo@UPorto, was implemented in Portugal during 2018 through University of Porto as a partner of the global Citizen Science project 'Tiny Earth' responding to the AMR crisis. In this first edition of MicroMundo@UPorto, university students (n = 41; Pharmaceutical Sciences and Nutrition Sciences) organized in eight teams tutored by university professors/researchers (n = 13) on Microbiology and AMR theoretical and practical aspects as well on communication skills to enable their guidance of younger school students (n = 140/3 schools) in experiments to discover antimicrobial-producing microorganisms while exploring the soil microbial diversity. Post-survey-based evaluation revealed that this project allowed university students to acquire diverse personal, social and scientific skills while increasing AMR awareness, in the One-Health perspective, and interest for science in school students. This University to Society approach can be successfully extended across Portugal and for education in Microbiology in general, with benefits for the future generations contributing to socially responsible and scientifically-literate citizens.

Lactobacillus mulieris sp. nov., a new species of Lactobacillus delbrueckii group.

One Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, and coccobacilli-shaped strain, designated c10Ua161MT, was isolated from a urine sample from a reproductive-age healthy woman. Comparative 16S rRNA gene sequence analysis indicated that strain c10Ua161MT belonged to the genus Lactobacillus. Phylogenetic analysis based on pheS and rpoA gene sequences strongly supported a clade encompassing strains c10Ua161MT and eight other strains from public databases, distinct from currently recognized species of the genus Lactobacillus. In silico Average Nucleotide Identity (ANI) and Genome-to-Genome Distance Calculator (GGDC), showed 87.9 and 34.3 % identity to the closest relative Lactobacillus jensenii, respectively. The major fatty acids of strain c10Ua161MT were C18 : 1ω9c (65.0%), C16 : 0 (17.8%), and summed feature 8 (10.2 %; comprising C18 : 1ω7c, and/or C18 : 1ω6c). The DNA G+C content of the strains is 34.2 mol%. On the basis of data presented here, strain c10Ua161MT represents a novel species of the genus Lactobacillus, for which the name Lactobacillus mulieris sp. nov. is proposed. The type strain is c10Ua161MT (=CECT 9755T=DSM 108704T).

Citrobacter europaeus sp. nov., isolated from water and human faecal samples.

Strains 97/79T and A121, recovered respectively from human faeces and well water, were compared to currently known species of the genus Citrobacter using genotypic and phenotypic approaches. Multilocus sequence analysis based on housekeeping genes fusA, leuS, pyrG, rpoB and recN, showed that the two strains formed a distinct phylogenetic lineage within the genus Citrobacter. Average nucleotide identity (ANI) between strains 97/79T and A121 was 99.2 %, whereas ANI values of strain 97/79T with the type strains of closely related species of the genus Citrobacter, C. werkmanii, C. braakii, C. freundii, C. youngae and C. pasteurii, were all below 93.0 %. The ability to metabolize different compounds also discriminated strains 97/79T and A121 from other species of the genus Citrobacter. Based on these results, strains 97/79T and A121 represent a novel species of the genus Citrobacter, for which the name Citrobacter europaeus sp. nov. is proposed, with strain 97/79T (=CIP 106467T=DSM 103031T) as the type strain. The DNA G+C content of strain 97/79T is 52.0 %.

Phylogeny and Comparative Genomics Unveil Independent Diversification Trajectories of qnrB and Genetic Platforms within Particular Citrobacter Species.

To gain insights into the diversification trajectories of qnrB genes, a phylogenetic and comparative genomics analysis of these genes and their surrounding genetic sequences was performed. For this purpose, Citrobacter sp. isolates (n = 21) and genome or plasmid sequences (n = 56) available in public databases harboring complete or truncated qnrB genes were analyzed. Citrobacter species identification was performed by phylogenetic analysis of different genotypic markers. The clonal relatedness among isolates, the location of qnrB genes, and the genetic surroundings of qnrB genes were investigated by pulsed-field gel electrophoresis (PFGE), S1-/I-CeuI-PFGE and hybridization, and PCR mapping and sequencing, respectively. Identification of Citrobacter isolates was achieved using leuS and recN gene sequences, and isolates characterized in this study were diverse and harbored chromosomal qnrB genes. Phylogenetic analysis of all known qnrB genes revealed seven main clusters and two branches, with most of them included in two clusters. Specific platforms (comprising pspF and sapA and varying in synteny and/or identity of other genes and intergenic regions) were associated with each one of these qnrB clusters, and the reliable identification of all Citrobacter isolates revealed that each platform evolved in different recognizable (Citrobacter freundii, C. braakii, C. werkmanii, and C. pasteurii) and putatively new species. A high identity was observed between some of the platforms identified in the chromosome of Citrobacter spp. and in different plasmids of Enterobacteriaceae. Our data corroborate Citrobacter as the origin of qnrB and further suggest divergent evolution of closely related qnrB genes/platforms in particular Citrobacter spp., which were delineated using particular genotypic markers.