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A hypothesis-forming exploratory cross-sectional assessment was conducted to assess the occurrence and relevance of Gram-positive rod-shaped bacteria like Corynebacterium spp. and Actinomycetaceae in human urine samples. In total, 1170 urine samples from 1031 inpatients with suspected urinary tract infection were assessed for culture-based growth of Gram-positive rod-shaped bacteria applying API Coryne assays, matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and in-house 16S rRNA gene sequencing. Overall, 502 different bacterial colonies from 346 urine samples taken from 324 inpatients were observed. The three quantitatively most abundant genera or genus clusters were Corynebacterium (254 isolates, 62%), Actinomyces/Winkia (79 isolates, 19%), and Actinotignum/Actinobaculum (29 isolates, 7%). Compared to sequencing, the diagnostic accuracy of all assessed competitor assays from the diagnostic routine was <80% for differentiation on the genus level and <30% for differentiation on the species level. Prolongated incubation for 4 days compared to 2 days resulted in additional detection of 15% of the totally recorded Gram-positive rod-shaped bacteria. An approximately 5-fold increased detection rate in mid-stream urine compared to urine acquired applying alternative sampling strategies was observed. In conclusion, in the rare event of the suspected clinical relevance of such findings, confirmatory testing with invasively sampled urine should be considered due to the high contamination rate observed in mid-stream urine. Confirmatory testing by DNA-sequencing methods should be considered if an exact identification of genus or species is regarded as relevant for the individual choice of the therapeutic strategy. Full article

(1) Background: Actinotignum schaalii is an emerging, opportunistic pathogen often linked with UTIs but can extend beyond the urogenital system. Data on the clinical significance of A. schaalii are still emerging. (2) Methods: A retrospective review of A. schaalii isolates in a Canadian tertiary care hospital was conducted. The clinical data of patients that grew A. schaalii from January 2020 to 2022 were documented. Demographics, site, management, and microbiological parameters surrounding culture and sensitivities were recorded. (3) Results: A total of 43 cases of A. schaalii were identified. Actinotignum schaalii was primarily involved in UTIs (n = 17), abscesses (n = 9), bacteremia (n = 6), septic arthritis (n = 5), and ulcers (n = 5). A. schaalii had a slight predilection for polymicrobial infections (51.1%, n = 22 out of 43), with Aerococcus urinae (n = 5) being the most common coisolate. Susceptibility testing was only performed in two cases that showed sensitivity to beta-lactam antibiotics and resistance to metronidazole and ciprofloxacin. Amoxicillin–clavulanate (n = 5) is the most frequently prescribed antibiotic. (4) Conclusions: The non-urogenic clinical significance of A. schaalii remains undervalued. The management of A. schaalii infection is multimodal, consisting predominantly of antimicrobials and surgical procedures specific to the etiology. Clinicians should request sensitivities for A. schaalii so that appropriate antimicrobial coverage can be provided. Full article

Actinotignum schaalii is an emerging, opportunistic pathogen and its connection to non-infectious diseases and conditions, such as prostate or bladder cancer, or chronic inflammation has been proposed. Here, we analyzed 297 urine, ureteral and urinary catheter samples from 128 patients by Polymerase Chain Reaction followed by Denaturing Gradient Gel Electrophoresis and Sequencing (PCR-DGGE-S), and culture, and 29 of these samples also by 16S rRNA Illumina sequencing, to establish A. schaalii’s prevalence in urinary tract-related samples, its relation to other bacteria, and its potential association with patients’ conditions and samples’ characteristics. A. schaalii-positive samples were significantly more diverse than A. schaalii negative and between-group diversity was higher than intra-group. Propionimicrobium lymphophilum, Fusobacterium nucleatum, Veillonella sp., Morganella sp., and Aerococcus sp. were significantly more often present in A. schaalii-positive samples; thus, we suggest these species are A. schaalii’s concomitants, while Enterobacter and Staphylococcaceae were more often identified in A. schaalii-negative samples; therefore, we propose A. schaalii and these species are mutually exclusive. Additionally, a significantly higher A. schaalii prevalence in patients with ureter stricture associated hydronephrosis (p = 0.020) was noted. We suggest that A. schaalii could be an early polybacterial biofilm colonizer, together with concomitant species, known for pro-inflammatory features. Full article

Actinobaculum massiliense, a Gram-positive anaerobic coccoid rod colonizing the human urinary tract, belongs to the taxonomic class of Actinobacteria. We identified A. massiliense as a cohabitant of urethral catheter biofilms (CB). The CBs also harbored more common uropathogens, such as Proteus mirabilis and Aerococcus urinae, supporting the notion that A. massiliense is adapted to a life style in polymicrobial biofilms. We isolated a clinical strain from a blood agar colony and used 16S rRNA gene sequencing and shotgun proteomics to confirm its identity as A. massiliense. We characterized this species by quantitatively comparing the bacterial proteome derived from in vitro growth with that of four clinical samples. The functional relevance of proteins with emphasis on nutrient import and the response to hostile host conditions, showing evidence of neutrophil infiltration, was analyzed. Two putative subtilisin-like proteases and a heme/oligopeptide transporter were abundant in vivo and are likely important for survival and fitness in the biofilm. Proteins facilitating uptake of xylose/glucuronate and oligopeptides, also highly expressed in vivo, may feed metabolites into mixed acid fermentation and peptidolysis pathways, respectively, to generate energy. A polyketide synthase predicted to generate a secondary metabolite that interacts with either the human host or co-colonizing microbes was also identified. The product of the PKS enzyme may contribute to A. massiliense fitness and persistence in the CBs. Full article