Karen C. Carroll

Knowledge of novel and revised bacterial taxonomy facilitates routine operations in the medical microbiology laboratory and communication with important stakeholders. Notable new gram-positive taxa accepted in 2024 include the clinically significant Staphylococcus brunensis sp. nov., as well as Streptococcus suis subsp. hashimotonensis subsp. nov., which can be delineated from other Streptococcus suis subsp. by possession of the Lancefield group A antigen. Four novel Enterobacterales species are discussed, plus a novel eighth family Gallaecimonadaceae . One of these, Providencia huashanensis sp. nov. is clinically significant and harbors multiple genetic determinants that encode antimicrobial resistance mechanisms. Stenotrophomonas forensis sp. nov., derived from specimen transport medium, was determined to be a distinct component within the Stenotrophomonas maltophilia complex. Bartonella tamiae sp. nov. has become officially recognized as the agent of the first culture-confirmed bartonelloses in Thailand. Significant taxonomic revisions include the return of Chlamydophila caviae to the Chlamydia genus, creation of the novel Metaclostridioides genus for Clostridioides mangenotii , and reclassification of two Kocuria spp. into two subspecies of Kocuria rosea . Clinical updates are provided for 13 past Journal of Clinical Microbiology taxonomic compendium entries for which extensive clinical data were not previously available. These include potential clinical significance for non- vaginalis species of the Gardnerella genus and for Pandoraea commovens in a non-cystic fibrosis setting.

C. difficile and the 3728T slurry induce rapid tumorigenesis in GF ApcMin/+ mice despite representing only a fraction of the luminal and mucosal microbiota. A and B, Twenty-five GF ApcMin/+ mice were gavaged with the 3728T slurry; five mice were sacrificed per time point. A, Colonic microadenoma and adenoma counts. B, Hematoxylin and eosin (H&E) staining of a representative microadenoma is shown from a mouse at 2 weeks p.i. (top), whereas a large adenoma coated by a dense BF is shown from a mouse at 4 weeks p.i. (bottom). Confocal imaging of Carnoy's-fixed mouse distal colons stained with the EUB338 universal probe (magenta), Cd198 C. difficile probe (yellow), or DAPI counterstain (blue). White arrows highlight individual C. difficile rods. C and D, Microadenomas (C) and FISH staining (D) of 2 weeks p.i. distal colons from GF ApcMin/+ mice gavaged with the 3728T isolates ± C. difficile CIm_3728T. Crosses represent mouse deaths. n = 6 mice per group, from two independent experiments. FISH images represent three separate mice per inoculum. White arrowheads denote areas with occasional mucus-invasive BFs directly interacting with epithelium in mice gavaged with the 3728T isolates without C. difficile (3728T isos – Cd). White arrows (bottom) represent areas with crypt-invasive BFs in the mice gavaged with the 3728T isolates + C. difficile. Statistical significance in A and C was calculated via Kruskal–Wallis followed by Mann–Whitney tests. Mann–Whitney P values are shown. E, Relative abundance (RA) of C. difficile derived from 16S rRNA amplicon sequencing of the original human slurry (3728T) or mouse stool obtained at sacrifice of mice in the time course experiment following gavage with the 3728T slurry. dpi, days post-inoculation. F, FISH of the original patient tumor 3728T. CRC, colorectal cancer. G–M, scRNA-seq and gene set enrichment analysis of 3728T slurry (n = 3)– versus 3979T slurry (n = 3)–gavaged GF ApcMin/+ mice at 2 weeks p.i. Heat maps depict genes enriched in canonical Wnt (G) or ROS (H) signaling pathways, with Wilcoxon rank-sum test P values above each gene. I, Inflammatory signaling pathways enriched in specific cell types from 3728T slurry– versus 3979T slurry–gavaged mice at 2 weeks p.i. KEGG, Kyoto Encyclopedia of Genes and Genomes. J, Inhibition and activation of the most significant upstream regulators in either progenitor or differentiated colonocytes. Expression log ratio is an effect size statistic. K and L, Network analysis of transcription factors from IPA upstream regulators enriched in the progenitor cell gene list revealed activation of Wnt/Myc signaling and proliferation, respectively. Lines connect transcription factors with shared target genes in the gene list. Darker lines indicate a greater number of shared target genes. M, Similar network analysis for differentiated colonocytes showed activation of the innate immune response.

C. difficile–associated tumorigenesis is toxin B (TcdB)–dependent. A, GF ApcMin/+ mice were gavaged with WT or isogenic tcdA−, tcdB−, or tcdA−tcdB− TargeTron mutant strains of M7404, an epidemic strain of ribotype 027, either simultaneously with the 3728T isolates or 1 week later. Tumor counts at 8 weeks p.i. were analyzed. Crosses represent mouse deaths. n = 2–7 mice per group, from two independent experiments. B, Summary graph showing colonic tumor induction of all TcdB− versus TcdB+ M7404 strains in combination with the 3728T isolates in GF ApcMin/+ mice. C and D,C. difficile stool CFUs and Vero toxin titers. Mann–Whitney P value is shown. E–M, Experimental design for 2-week experiments with M7404 strains: mice were gavaged with the 3728T isolates − Cd 1 week prior to C. difficile gavage with either the tcdB− (n = 6 mice) or WT M7404 strain (n = 8 mice). F and G,C. difficile stool CFUs and toxin titers. H, Representative crypts with BF invasion, stained in red for EUB338 and yellow for Cd198. I,C. difficile tissue invasion. J, Representative H&E images and quantitation of histopathologic changes at 2 weeks p.i. K, Representative images of β-catenin, Ki-67, and PCNA immunofluorescent staining of distal colonic epithelium in M7404 TcdB− (top)– versus M7404 WT (bottom)–inoculated mice. Arrow denotes a dysplastic focus in the M7404 WT–inoculated mouse. L, Quantitation and representative images of crypt depth in the distal colon. M, Host gene expression in colon. Dashed line represents 3728T isolates − Cd-negative control. Statistics shown are Mann–Whitney t tests. ns, not significant.

C. difficile induces the activation of myeloid cells and IL17-producing lymphoid subsets. Multiparameter flow cytometry analysis of colonic lamina propria innate (myeloid and ILCs) and adaptive immune cell populations of LPL isolated from colons collected at 2 weeks p.i. from GF ApcMin/+ mice gavaged with the 3728T isolates with (n = 5) or without (n = 6) C. difficile strain CIm_3728T. Results from two independent experiments. A, Opt-SNE projection of myeloid and ILC populations. B, Dot plots of myeloid cell type abundance as a percentage of the CD45 population. C, Representative gating of DCs, PMN, and IL17 expression by ILC3 cells. D, Opt-SNE projection of adaptive populations (B and T lymphocytes). E, Dot plots of changes in abundance of lymphoid populations with C. difficile. F, Representative gating of lymphoid subsets. Opt-SNE projections are used for visualization purposes only. Statistical analyses in B and E represent Mann–Whitney t tests based on the gating in C and F, respectively. DC, dendritic cells; EILP, early innate lymphoid progenitors; ILC, innate lymphoid cells; IMC, innate myeloid cells; LPL, lamina propria leukocytes; MAC, macrophages; MO, monocytic cells; NK, natural killer; PMN, polymorphonuclear cells; Treg, regulatory T cells.

C. difficile strains isolated from human colon cancer mucosa drive colonic tumor formation in GF ApcMin/+ mice. A and B, Variation in colonic tumorigenic potential in GF ApcMin/+ mice at 12 to 14 weeks post-inoculation (p.i.) by individual BF-positive tumor (BF+T; A) or BF-negative tumor (BF−T; B) colorectal cancer mucosal samples from six patients. n = 6–8 mice per group, from two independent experiments. C, 16S rRNA amplicon sequencing relative abundances of the top 20 species in the six patient tumor slurry inocula and 12 to 14 weeks p.i. stools from mice gavaged with the tumor slurries. D, A consortium of 30 bacterial isolates derived from GF mice gavaged with the 3728T slurry recapitulated colonic tumorigenesis of the original 3728T slurry. n = 5–11 mice per group. E and F, Colonic tumor counts (E) and representative methylene blue staining (F) of colonic tumors (arrows) at 12 weeks p.i. in a vancomycin/gentamicin model (see Methods) of chronic colonization with nontoxigenic (TcdA−TcdB−) or toxigenic (TcdA+TcdB+) C. difficile strains. n = 5–18 mice per group, from two independent experiments. G and H, Colonic tumor counts (G) and representative methylene blue staining (H) of colonic tumors (arrows) from GF ApcMin/+ mice gavaged with the 3728T isolates with or without the CIm_3728T or CIm_3752T C. difficile strains. n = 6–8 mice per group. I, Colonic tumors induced in GF ApcMin/+ mice gavaged with either the 3979T slurry or the 3979T slurry with C. difficile strain CIm_3728T at 10 weeks p.i. The cross represents a mouse death. n = 7–8 mice per group. Statistical significance was calculated via Kruskal–Wallis followed by Mann–Whitney tests. Mann–Whitney P values are shown. ns, not significant.

Clinical and Laboratory Standards Institute (CLSI) recommends a blood culture contamination (BCC) threshold of <3%, with ≤1% considered optimal. However, there is not a standardized definition of BCC, and the effect of multiple definitions on BCC rates or what definitions laboratories use remain unknown. We surveyed 52 hospitals and analyzed 362,078 blood cultures (BCx) collected 1 September 2019 to 31 August 2021 from 62 intensive care units (ICUs) and 231 wards from 48 of these hospitals. We calculated and compared BCC rates using the College of American Pathologists (CAP) or CLSI criteria (both utilize a limited number of skin commensals to define BCC) and the comprehensive National Healthcare Safety Network (NHSN) commensal list. We characterized factors associated with BCC and related outcomes (central-line associated bloodstream infection [CLABSI] and vancomycin use). BCC, BCx positivity, and single BCx rates were monitored by 100%, 39%, and 21% of hospitals, respectively. Hospitals used CAP (65%), CLSI (17%), and NHSN (17%) criteria to define BCC. Mean BCC rate by CAP (CAP-BCC) was 1.38% for ICUs and 0.96% for wards. BCC rates remained similar by CLSI criteria but increased when using NHSN list. Sharing BCC data outside of the laboratory, measuring additional BCx quality indicators, and limiting central catheter-drawn BCx were associated with lower BCC rates. BCC was associated with higher CLABSI rates in ICUs. This study demonstrated variability in laboratory practices and opportunities to optimize BCx stewardship. IMPORTANCE: Blood culture contamination (BCC) is associated with patient harm and unnecessary use of healthcare resources. BCC thresholds have been established; however, multiple BCC definitions exist. There is limited data on how BCC rates differ depending on the BCC definition used, what definitions laboratories most commonly use, or their approach to other blood cultures (BCx) quality indicators such as single rates or BCx positivity. A cross-sectional multicenter survey and analysis of BCx data from intensive care unit and wards revealed that most laboratories did not track single BCx or BCx positivity rates and that there was variability in how BCC was defined. Additionally, BCC rates were influenced by the definition used. BCC was associated with increased central-line associated bloodstream infection rates.

Sanger sequencing of the first ~500 bp of the 16S rRNA gene is frequently used to identify bacterial pathogens that have ambiguous biochemical profiles or proteomic mass spectra. When diversity does not occur within that region, genus-level and/or species-level identification may not be possible, and a longer sequence or alternative target may be required to distinguish between genera/species. In this study, we evaluated a clinically relevant end-to-end solution for long-read (~1,500 nt) 16S rRNA next-generation sequencing by Oxford Nanopore Technologies (ONT) compared to a ~500 nt Sanger sequencing approach for the identification of 153 bacterial clinical isolates. Sequencing data were analyzed using the IDNS software from SmartGene and its proprietary 16S Centroid reference database (Centroid database) SmartGene software and the Centroid database. The agreement of the two platforms on species- and genus-level identification was determined, and discrepancies were resolved by whole-genome sequencing. ONT had a higher taxonomic resolution at the genus level ( P < 0.01). When genus-level identification was achieved by both methods, concordance to the best matching genus was 100%. When species-level identification was achieved by both methods, concordance to the best matching species was 91%. The costs per test were ~$25.30 (when multiplexing 24 samples/run) and $74 for ONT and Sanger sequencing, respectively. The hands-on time spent performing sequencing was similar for both methods, but the turnaround time of ONT was significantly shorter than that of Sanger sequencing. IMPORTANCE: This study adds to existing literature by describing a validated end-to-end solution of 16S rRNA gene Oxford Nanopore sequencing for bacterial isolate identification, including sequencing run time evaluation, automated analysis (SmartGene 16S Identification App) and interpretation of results, that can be incorporated into clinical and public health laboratories with a simple and cost-effective workflow.

Taxonomy is a systematic practice in which microorganisms are granted names to facilitate and standardize multi-disciplinary communication. We summarize novel bacterial taxa derived from human clinical material that were published in peer-reviewed literature and/or included by the International Journal of Systematic and Evolutionary Microbiology during calendar year 2023, as well as taxonomic revisions that have been published/included by the same entity. While the majority of newly discovered facultative and anaerobic organisms were derived from microbiome surveillance, noteworthy novel taxa in the realm of pathogenicity potential include those related to Aerococcus spp., several Corynebacterium spp., Exercitatus varius gen. nov., sp. nov., and Mycoplasma phocimorsus sp. nov. With respect to nomenclature revision, the Bacillus and Clostridium genera continue to be visited annually. Creation of novel anaerobic Gram-negative bacillus genera Hallella , Hoylesella , Leyella , Segatella , and Xylanibacter impacted several Bacteroides spp. and Prevotella spp. Additional studies are necessary to ascertain the clinical significance of several of these microbes.