Frontiers in Cellular and Infection Microbiology

The Gardnerella genus includes a complex sub-taxonomy of bacterial species and subgroups which have been implicated in bacterial vaginosis. We analyzed G. leopoldii, G. swidsinski, G. piotii, and G. vaginalis genomes available in public databases and predicted proviral sequences using Cenote-Taker3. We clustered 266 prophage sequences into 140 viral genera level groupings that encode four distinct gene clusters and 19 different lysin proteins. Analysis of the bacterial and prophage sequence phylogenies reveals that while there were no specific prophage sequence lineages that infected each bacterial host, there is clustering of the prophage sequences by bacterial species within the Gardnerella genus.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, the most common curable non-viral sexually transmitted infection, and Chlamydia trachomatis is a bacterial pathogen that can ascend to the upper genital tract and cause pelvic inflammatory disease, infertility, and ectopic pregnancy. T. vaginalis harbors bacterial endosymbionts, including Candidatus Malacoplasma girerdii, an obligate symbiont, and Metamycoplasma hominis, which can live freely or symbiotically. In a 16S rRNA sequencing study of the cervicovaginal microbiome of women at high risk for chlamydial infection, Ca. M. girerdii abundance was one of 13 features predicting lack of chlamydial spread to the endometrium, despite no direct association between T. vaginalis infection and reduced chlamydial ascension. Investigating the relationship between these microorganisms further, we found that T. vaginalis vaginal abundance correlated positively with chlamydial burden in women whose infection was confined to the cervix, while a nonsignificant inverse relationship was seen in women with endometrial spread. Among participants with high chlamydial burden, Ca. M. girerdii was detected exclusively in women without endometrial infection. Both endosymbionts trended toward more frequent detection, and higher abundance, in coinfected women without endometrial spread, while M. hominis abundance correlated strongly with T. vaginalis burden in this group. These findings suggest that mycoplasmal endosymbionts of T. vaginalis, rather than T. vaginalis itself, are microbial factors limiting chlamydial ascension, and point to a three-way interaction between parasite, endosymbiont, and bacterial pathogen that shapes upper genital tract C. trachomatis infection risk.

ABSTRACT Background: Trachoma remains the leading infectious cause of blindness. Over 100M people are at risk, and 1.9M already live with visual impairment or blindness. Ethiopia bears the greatest burden of disease with ~61M or 59% of the known affected global population. Although Chlamydia trachomatis (Ct) infection initiates disease, infection alone fails to explain why inflammation resolves in some but progresses to irreversible scarring and vision loss in others. Previous studies have largely examined gene expression or limited protein panels, leaving the conjunctival immune protein environment poorly characterized. We hypothesized that distinct conjunctival immune states capture these pathogenic processes and could reveal biomarkers for selecting treatment and surveillance strategies. Methods and Findings: We conducted a cross-sectional study of 667 participants residing in the hyperendemic Amhara region of Ethiopia at least one year after the most recent azithromycin mass drug administration to treat Ct. The upper tarsal conjunctivae was graded for TF and TI, trachomatous inflammation-follicular or intense, respectively; I-TS/TT, inflammation with scarring with or without trichiasis, defined as one or more eyelashes touching the globe of the eye; and TS and TT, scarring with or without trichiasis but no inflammation. T0 represented no trachoma. The protein concentrations of 41 immune mediators were quantified using Meso Scale Discovery electrochemiluminescence immunoassays. Associations with infection and grade were evaluated using covariate-adjusted models with false discovery rate correction. Bernoulli mixture modeling (BMM) revealed four distinct Immune State Types (ISTs). Both approaches supported a shared biological framework characterized by selective immune activation in Ct infection, broad inflammation in TF/TI, and chemokine remodeling versus immune quiescence in the scarred conjunctiva. Ct infection was associated with a focused IFN-{gamma}-centered response. IST1 captured this immune response where 51.9% of the participants had Ct; we therefore termed IST1 Quiescent Infection. In contrast, TF/TI was associated with higher concentrations of proinflammatory, Th1/Th17, chemokine, regulatory, and remodeling pathways mediators. This profile fit well within IST2, termed Broad Inflammation, where 72.4% of the participants had TF/TI. TS/TT retained considerable chemokine activity, whereas I-TS/TT showed a narrower, IL-17A-dominant inflammatory profile. BMM separated scarring into two states characterized by distinct profiles: IST3, Conjunctival Remodeling, with significantly stronger chemokine and T-cell maintenance signals; and IST4, Quiescent Scarring, with the majority of chemokines being significantly downregulated. This suggests that scarred conjunctiva can retain substantial immune activity or shift toward a more attenuated state. Conclusions: This study establishes the first IST framework for trachoma, revealing biologically distinct immune states with different potential clinical implications. IST1 indicates the need for antibiotic treatment, whereas IST2 suggests that novel immunotherapeutics might be effective in thwarting disease progression. The upregulated chemokines in IST3 may indicate risk of progression to TT necessitating prospective studies and potential novel antifibrotic interventions. Together, our findings provide a strong biological foundation for stratifying patients by immune state and matching them to preventive and therapeutic care. Prospective validation and streamlined biomarker panels will support the translation of this framework into field settings to help avert disease progression and blindness.

The fungal pathogen Cryptococcus neoformans contains approximately 200 {micro}g of {beta}-1,3-glucan (1,3BG) per 1 mg of dry cell weight when grown under standard culture conditions (YPD medium at 30{degrees}C under aerobic conditions). However, 1,3BG exposure is tightly suppressed, even in capsule-deficient strains, allowing the fungus to evade recognition by the immune receptor dectin-1 and anti-1,3BG antibodies. Although other pathogenic fungi mask 1,3BG with -1,3-glucan (1,3AG) to evade dectin-1 recognition, the factors responsible for 1,3BG masking and dectin-1 evasion in C. neoformans remain incompletely understood. To identify capsule-independent 1,3BG masking and dectin-1 evasion factors, we generated a series of cell wall-related gene deletion strains in the capsule-deficient strain cap59{Delta} using CRISPR/Cas9 and screened for mutants that failed to evade dectin-1 binding. We found eight deletants (cap59{Delta}/mpk1{Delta}, cap59{Delta}/chs3{Delta}, cap59{Delta}/kre5{Delta}, cap59{Delta}/crz1{Delta}, cap59{Delta}/kre6{Delta}/skn1{Delta}, cap59{Delta}/hxl1{Delta}, cap59{Delta}/uge1{Delta}, and cap59{Delta}/ugt1{Delta}) that exhibited increased binding to dectin-1 and/or anti-1,3BG antibody. Since a similar phenotype was not observed in cap59{Delta}/ags1{Delta}, 1,3AG-mediated masking of 1,3BG appears to play a limited role in C. neoformans. These eight deletants induced significantly greater secretion of IL-6 and IL-1{beta} from dendritic cells (DCs) than cap59{Delta} or cap59{Delta}/ags1{Delta}. This enhanced inflammatory response was markedly attenuated in dectin-1-deficient DCs, indicating that the increased immunogenicity was driven by 1,3BG exposure and subsequent dectin-1 recognition. Collectively, these findings demonstrate that multiple genes involved in maintaining cell wall integrity, including those involved in {beta}-1,6-glucan and chitosan biosynthesis, are essential for regulating 1,3BG exposure and enabling C. neoformans to evade dectin-1-mediated immune recognition. HighlightsO_LINovel capsule-independent {beta}-1,3-glucan masking genes in Cryptococcus neoformans were identified. C_LIO_LIThe deletants of these genes displayed higher dectin-1 deposition, contrary to the parental capsule-deficient mutant cap59{Delta}. C_LIO_LIDeletion of these genes led to enhanced secretion of IL-6 and IL-1{beta} from dendritic cells. C_LIO_LIThe enhanced cytokine response was suppressed in dendritic cells lacking dectin-1. C_LIO_LIThese deletant strains have potential to serve as new whole-cell antigens for cryptococcal vaccine development. C_LI

Orientia tsutsugamushi is the causative agent of scrub typhus infection in humans. This pathogen is transmitted into mammalian hosts including humans through Leptotrombidium mites. Till date, detection of O. tsutsugamushi in human was performed using PCR assay with 56kDa type specific antigen (TSA56), 47kDa outer membrane protein (47kDa-OMP) and groEL chaperonin gene. To understand the efficiency of different genes for the detection of O. tsutsugamushi in Leptotrombidium mites, we performed a comparative PCR assay with various universal primers targeting different genes including TSA56, 47kDa-OMP and groEL gene. We have collected 500 Leptotrombidium mites from Vellore, Tamil Nadu and, the mites were segregated into 50 pools with 10 mites in each pool were used for DNA extraction. The molecular detection of O. tsutsugamushi was performed using PCR assays (including conventional, nested and duplex PCR). The comparative PCR analysis showed nested PCR of groEL gene has high efficiency in detecting the presence of O. tsutsugamushi in chigger samples compared to other genes. Interestingly, groEL target demonstrated high efficiency for detection of O. tsutsugamushi in chigger samples and, when used with nested primers, can facilitate differentiation of O. tsutsugamushi from other rickettsial pathogens. Thus, we conclude groEL gene nested PCR assay can be used routinely to detect O. tsutsugamushi in trombiculid mites.

Background: European Association of Urology (EAU) Guidelines identify urogenital infections as a possible treatable cause of infertility, yet the current recommendation for antibiotic treatment remains weak due to a lack of high-quality evidence. We evaluated the effect of antibiotic treatment of asymptomatic urogenital infections on both conventional and apoptosis-related (flow cytometry sperm apoptosis test, FACS-APO) sperm parameters, as well as on pregnancy outcomes, in infertile couples. Methods: In this retrospective, single-centre study, we analysed 265 couples with an asymptomatic urogenital infection (Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, and Chlamydia trachomatis) confirmed in at least one partner by first-void urine testing, who received simultaneous antibiotic treatment. Paired conventional semen analysis and FACS-APO were performed before and 0-9 months after treatment completion. Partner infection concordance, the effect of follow-up timing on treatment response, and pregnancy outcomes within 12 months were also assessed. Results: Sperm concentration, total sperm count, total and progressive motility, and the proportion of viable sperm increased significantly after treatment, while the proportion of late apoptotic / necrotic sperm and calculated sperm DNA fragmentation decreased significantly (all p<0.05); the proportion of early apoptotic sperm remained unchanged, and semen volume did not change. Among 244 couples with complete bilateral microbiological testing, Ureaplasma urealyticum was by far the most prevalent pathogen (57.8% of men, 87.7% of women), and female partner positivity given male positivity was 83.0%. The improvement in sperm concentration and total sperm count was most pronounced within 3 months after treatment and diminished, or reversed, beyond 6 months. Pregnancy was achieved in 127 of 265 couples (47.9%; per-protocol rate 53.4%) within 12 months, most commonly following in vitro fertilization (72/127) or spontaneously (21/127). Conclusions: Antibiotic treatment of asymptomatic urogenital infections in infertile couples was associated with significant improvement in conventional semen parameters and a reduction in sperm apoptosis and calculated DNA fragmentation. Given the retrospective, uncontrolled design, these findings should be regarded as hypothesis-generating rather than definitive proof of a causal, fertility-enhancing effect, and warrant confirmation in prospective controlled studies.

Adherent Invasive Escherichia coli (AIEC) colonize the ileal mucosa of Crohn's disease patients. Among bacterial virulence factors potentially implicated, type 6 secretion systems (T6SS) are molecular machineries sending effectors in prokaryotic or eukaryotic target cells. Two T6SS have been reported in AIEC LF82 reference strain. Here, we show that T6SS genes are more prevalent in Crohn's disease patients' microbiota compared to healthy controls, and that T6SS are more frequent in AIEC pathovar compared to E. coli in general and associated with FimH pathoadaptive polymorphism. The use of an in vitro gastrointestinal system coupled with T6SS promoter reporter assay highlighted expression of LF82 T6SS under gastrointestinal conditions, with a major role for bile salts. In mice infected with AIEC LF82 versus T6SS-deleted mutants, T6SS showed a functional impact in modulating the composition of mucosa-associated gut microbiota. Thus, AIEC may use their T6SS to compete with endogenous microbiota to colonize host mucosa.

Despite increased recognition of the diverse resident microbiome of the urinary tract (i.e., the urobiome) in postmenopausal women, the roles and functions of these microbes remain largely unknown. Further empirical research is needed to understand the physiology, interactions, and antibiotic resistance evolution of urobiome members with pathogenic potential. However, experimental work relies on viable, culturable isolates. Standard urine culturing practices are designed for identifying a narrow set of known urinary microbes, and are thus poorly suited for cultivating taxa from the resident urobiome. Here we expand the urine culturing toolkit to reliably recover diverse urobiome taxa for downstream empirical research. Urine samples collected from postmenopausal women with recurrent urinary tract infections were shipped at ambient temperature to a central point for culturing. Microbial viability was maintained using boric acid preservative tubes during multi-day transport of sample aliquots. Selective media incubated under specialized conditions were used to promote recovery of diverse urobiome members, including fastidious taxa. Under 5% CO2 -enriched atmospheric conditions and with longer incubation times, we leveraged a chromogenic agar (UTIC) to further differentiate isolates based on colony color and morphology. We evaluated the workflow for its ability to isolate and characterize urobiome taxa, as determined by morphological differentiation and taxonomic identification. Across 108 urine samples, 6.3 {+/-} 3.2 distinct isolates were recovered, with no detectable relationship between sample shipment duration and isolate richness. On chromogenic agar, colony growth and color intensity was improved with CO2 -enriched atmospheric conditions and extended incubation times. We identified diverse taxa that are typically underrepresented in standard diagnostic culture and provide novel morphological characterizations for members of the genera Actinotignum, Aerococcus, Facklamia, Lactobacillus, Latilactobacillus, Limosilactobacillus, and Streptococcus species, which have not been previously described on UTIC chromogenic agar. Using this novel workflow, we recovered a diverse collection of urobiome isolates from urine samples shipped over multiple days. We also demonstrated the utility of a chromogenic agar for the visual differentiation of key urobiome taxa. While sequencing approaches have enhanced our understanding of urobiome composition, culturing is needed to investigate microbial interactions, virulence mechanisms, and antimicrobial susceptibility. This protocol adds to the growing toolkit for the cultivation of diverse urobiome isolates needed to support downstream empirical studies and advance urinary tract infection research.

Pre-prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first step in the lipoprotein modification pathway and is essential for growth and viability of Helicobacter pylori. We explored the fate of H. pylori cells under Lgt depletion conditions to obtain insight into Lgt function on morphology and physiology. Downregulation of lgt expression led to growth arrest and a heterogenous population of small rod-shaped cells and small round cells with spiky protrusions, a loose outer membrane and disintegrated flagella. H. pylori overcomes a defect in lipoprotein modification through alteration of its gene expression profile resulting in so-called revertants. Outer membrane protein synthesis, carbon metabolism and nutrient uptake was reduced, and protein translation and flagellar biogenesis increased. Lgt specific regulation was observed in phospholipid biosynthesis resulting in an initial increase in cardiolipin followed by an increase in lysophospholipids at the time of adaptation. Suppressors of {Delta}lgt that were selected on solid media under restrictive conditions either lacked chromosomal mutations or had mutations in the cagUT-tetR regulon controlling the expression of lgt. A suppressor without mutations had an altered expression profile that led to downregulation of a DNA methyltransferase and upregulation of HP_0014, a motility associated glycosyltransferase family protein. Transmission electron microscopy and single cell video microscopy showed that a subpopulation of cells is motile in the revertant while the suppressor regained full motility with higher average speed compared to the wild type strain. Overall, our data show that lipoprotein modification by Lgt is important for membrane and flagellar integrity and that the heterogenous character of H. pylori cultures allows individual cells to overcome this cell envelope stress and survive. Our findings are important for antibacterial development strategies targeting essential integral membrane enzymes involved in lipoprotein modification and stimulate exploration of synergetic activity between known antibiotics and novel antibacterial agents targeting novel cellular pathways.

Obligate endosymbionts relying on transovarial transmission must coordinate with host reproduction, yet the regulatory mechanisms remain poorly understood. Here we show that the vitellogenesis pathway of the tick Ixodes scapularis regulates the transcriptional state of its endosymbiont Rickettsia buchneri (Rb), separately from its abundance. In males, Rb DNA remained detectable, but bacterial transcription was strongly reduced across all examined genes, with markedly lower RNA/DNA ratios. In females, Rb was restricted to ovarian tissues (developing oocytes and interstitial cells), with no detection in salivary glands or midgut by TEM, FISH, or PCR. After blood feeding, Rb density within size-matched early-stage oocytes was significantly reduced. We further characterized two gene families mediating vitellogenesis: vitellogenin synthesis genes (Vgs, n = 20) and vitellogenin receptor genes (Vgr, n = 15). Vgs proteins showed conserved domain organization, whereas Vgr paralogs showed greater structural diversification. RNAi silencing of Vgs20 or Vgr12 altered Rb transcriptional profiles in both tick cells and ticks, although changes in bacterial load were not consistent between the two systems. Antibiotic depletion of Rb increased expression of both host genes. Together, these findings show that tick vitellogenesis pathways contribute to Rb regulation during reproduction and identify the I. scapularis-Rb system as a useful model for studying host control of obligate endosymbionts. IMPORTANCEMaternally inherited bacterial symbionts are commonly characterized by bacterial abundance. This study shows that bacterial abundance alone does not necessarily reflect symbiont functional state. In the blacklegged tick, the inherited symbiont Rickettsia buchneri persists in males but exhibits little transcriptional activity. In females, the host reproductive pathway determines whether the symbiont is active: silencing one component of this pathway changed bacterial gene expression without changing bacterial abundance. Hosts therefore regulate not only the abundance of inherited symbionts, but also their functional state. The same bacterial abundance can correspond to very different functional states. Understanding inherited symbioses therefore requires considering bacterial function alongside bacterial abundance.

Background Group B Streptococcus (GBS) is a leading cause of bacterial sepsis globally. Late-onset (LO) GBS disease is preceded by colonization within the gastrointestinal tract in vulnerable neonates. Previous studies demonstrate that Candida albicans (C. albicans) promotes GBS colonization of host tissues including the bladder and vagina. However, its impact on GBS colonization of the intestinal tract is unknown. Methods We established a new animal model to study GBS and C. albicans co-colonization model in the context of the newborn host to examine C. albicans impact on GBS and further evaluated fluconazole prophylactic treatment to these same outcomes. Physical interactions between organisms in vivo were imaged and GBS gene expression was evaluated. Results C. albicans increased GBS burden in the neonatal gut and GBS was physically colocalized with C. albicans hyphae as evidenced by the colocalization analysis. Fluconazole treatment of co-colonized mice decreased extraintestinal GBS dissemination. C. albicans presence upregulated genes encoding proteins responsible for GBS adhesion and invasion of intestinal epithelial cells. Conclusion C. albicans enhances GBS burden and pathogenicity in the neonatal gut. These findings highlight the need for further investigation into C. albicans as a potential risk factor for GBS colonization and LO disease in human infants.

Streptococcus anginosus (S. anginosus) has long been considered a commensal of the human microbiome but is increasingly associated with invasive infections and malignant processes. For understanding evolutionary dynamics, it is essential to investigate its mobile genetic elements, such as prophages, which are known to impact virulence, antibiotic resistance, and horizontal gene transfer. While many S. anginosus strains carry prophages, lysogen induction by external stimuli has not been demonstrated, and phage-mediated infection or lysis of this species has not been reported. To analyze the prevalence and diversity of prophages in S. anginosus genomes, we screened 140 clinical isolates by PCR revealing that 31.4% of strains were lysogenic. Correlating these findings with the presence of CRISPR immunity, we observed that S. anginosus strains carrying a CRISPR-Cas type II-A system were less likely to harbor prophages. Using a PCR-based approach, the spontaneous excision of several prophages of S. anginosus could be demonstrated and a fluoroquinolone-triggered prophage induction could successfully be established. Induction by ciprofloxacin and levofloxacin resulted in significant, concentration-dependent phage release and bacterial lysis. Transmission electron microscopy revealed viruses exhibiting the morphology characteristic of siphoviruses. Further analysis of the susceptibility of S. anginosus isolates and other oral and pyogenic streptococci to the isolated S. anginosus phages demonstrated a broad host range and the potential for cross-species horizontal gene transfer. In conclusion, a lytic cycle of S. anginosus phages could be induced, highlighting their functional relevance to pathogenicity and horizontal gene transfer, while demonstrating potential clinical implications of antibiotic-mediated prophage activation.

The Enterobacteriaceae pathogens like E. coli, Shigella, Klebsiella and Salmonella cause a wide range of gastrointestinal and other mucosal infections when the contaminated food and water are consumed. Conventional microbiological methods are time-consuming and laborious process to detect these pathogens. Molecular assays are fast and sensitive to detect the pathogens. But a potential target for the detection is very essential to develop a molecular assay for the rapid detection. In this study, we explored the cpn60 UT region as a potential target for the detection of significant Enterobacteriaceae members like E. coli, Shigella sp, Salmonella, Klebsiella etc. We did the phylogenetic analysis of cpn60 UT region among the Enterobacteriaceae pathogenic groups. This analysis has revealed the presence of 13 clusters and two oligonucleotide regions with highest nucleotide conservation. Targeting these regions we have designed a set of oligonucleotide primer pairs and developed a SYBR green real time PCR assay to detect the Enterobacteriaceae pathogenic members. The degenerate oligonucleotide primers designed amplified a 132 bp UT region of cpn60 gene present in Enterobacteriaceae pathogens. The sensitivity of the assay was initially investigated by 10-fold dilution series of overnight broth culture of E. coli ATCC 10536 and had a detection limit of 101 CFU/mL. The specificity was assessed by comparing the amplification plot of a set of Enterobacteriaceae pathogens and non-Enterobacteriaceae members like Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes) and found to detect all the Enterobacteriaceae cultures included in the study whereas amplification was not seen in S. aureus and L. monocytogenes.

Background Prediction and diagnosis of recurrent primary sclerosing cholangitis (rPSC) after liver transplantation remain challenging. We investigated anti-integrin v{beta}6 autoantibody titers and their association with rPSC development. Methods Twenty-three patients with PSC with serial serum samples and liver biopsies after liver transplantation were included. rPSC was diagnosed according to the 2024 Japanese diagnostic criteria. Anti-integrin v{beta}6 autoantibody titers were measured by enzyme-linked immunosorbent assays. Fold changes (FCs) in antibody titers were calculated relative to each patient's pre-transplant baseline, and the maximum FC during post-transplant follow-up was defined as FCmax. Results Antibody titers significantly decreased at the first postoperative measurement compared with the pre-transplant level. Ten of 23 patients developed rPSC. Three patients whose ulcerative colitis activity changed after transplantation were excluded from further analysis, leaving 20 patients. The maximum absolute antibody titer during post-transplant follow-up was not associated with rPSC development. However, FCmax was significantly higher in patients with rPSC than in those without rPSC (1.381 vs. 0.514, P = 0.041), with an AUC of 0.781. Conclusions Anti-integrin v{beta}6 autoantibody titers significantly decreased after liver transplantation for PSC. Post-transplant antibody re-elevation relative to the pre-transplant baseline was significantly associated with the development of rPSC.

Scrub typhus, caused by Orientia tsutsugamushi, is an obligate intracellular gram-negative pathogen that remains a cause of acute febrile illness in India. Culture isolation of Orientia tsutsugamushi clinical isolates is infrequent because it is technically more challenging than PCR-based molecular identification. In this report, we describe the culture isolation of Orientia from the whole blood of a 64-year-old farmer with acute febrile illness. Whole blood was inoculated onto an 80% confluent L929 cell line. Real-time PCR targeting the 47-kDa and 56-kDa genes, combined with Sanger sequencing, confirmed the isolate. Transmission electron microscopy of infected L929 cells revealed multiple oval-shaped bacteria within the host cytoplasm. Confocal microscopy demonstrated progressive accumulation of CFSE-labelled bacteria within infected cells over time. These findings support the successful isolation and visualization of a blood-derived O. tsutsugamushi isolate and provide a platform for downstream assays of host-pathogen interactions, antimicrobial susceptibility testing, and vaccine development.

Bloodstream infection (BSI) is a life-threatening condition where the risk of dying is affected by both patient- and pathogen-related factors. Certain microbial traits (e.g., virulence genes) are thought to affect survival rates from BSIs, but the magnitude of their impact on human mortality remains unclear. To address this, we analyzed the microbial genomes, quantitative proteomes, and electronic medical records (EMRs) from 35,746 BSIs from Calgary, Canada (2006-2022). We show that EMR-based machine learning models accurately predict 30-day mortality (AUC=0.83), but surprisingly, integrating >70,000 microbial genes/proteins does not improve this performance. We demonstrate that microbial factors co-segregate with specific patient cohorts and are therefore not independent predictors of risk. We conclude that microbial virulence factors have minimal impact on mortality rates from BSIs.

Chlamydia trachomatis is an obligate intracellular, developmentally regulated pathogen with a reduced genome as a consequence of adapting to the host cell environment. Despite possessing only eight transcription factors, its developmental cycle is tightly regulated. Hence, we hypothesize that factors like post-transcriptional and post-translational modifications play significant roles in controlling developmental progression. Chlamydia encodes serine/threonine kinases, including an ortholog of Pkn1. In this study, we characterized the function of Pkn1 in chlamydial biology and revealed its role as a cell division regulator. We designed an anhydrotetracycline (aTc)-inducible construct encoding Pkn1 tagged with six histidines at the C-terminus (Pkn1_6xH). Wild-type Pkn1 localized primarily at the division septum whereas a kinase-null Pkn1 isoform localized in the cytosol. To further explore the association of Pkn1 with the divisome, we designed a plasmid encoding an aTc-inducible CRISPRi system targeting pkn1(pkn1KD). Intriguingly, decreased pkn1 transcript levels resulted in enlarged chlamydial cells with irregular coccoid shapes akin to penicillin-treated cells, indicating a block in cell division when pkn1 transcripts are reduced. These effects were complemented by expressing an additional wild-type copy, but not a kinase-null copy, of pkn1 during knockdown. Using a bacterial two hybrid system, we identified an interaction between Pkn1 and MreB, a cytoskeletal protein forming a ring at the septum in Chlamydia. We showed the co-localization of these proteins in Chlamydia and that inhibition of MreB function disrupts Pkn1 localization. Overall, our data indicate an essential function for Pkn1 activity in regulating cell division in Chlamydia.

Aim: Brucellosis remains endemic throughout the Mediterranean basin, the Middle East and Central Asia. Genitourinary involvement is an uncommon but clinically consequential focal complication that closely mimics urological emergencies and may precipitate unnecessary surgery. Contemporary data describing its frequency, laboratory signature and treatment outcomes in endemic settings remain limited. Material-Methods: We retrospectively screened all adults ([&ge;]18 years) with confirmed brucellosis managed at a tertiary infectious diseases unit between January 2008 and December 2020. Genitourinary involvement was defined as epididymo-orchitis, orchitis, epididymitis and/or brucellar pyelonephritis. Clinical, laboratory, ultrasonographic, therapeutic and outcome variables were abstracted using a standardised form. Proportions are reported with Wilson score 95% confidence intervals (CI). Reporting followed the STROBE statement. Result: Genitourinary involvement was identified in 18 of 125 patients with brucellosis (14.4%; 95% CI 9.3-21.6); 16 (88.9%) were men and the mean age was 35.2 years (range 22-62). Scrotal disease dominated the male presentation: epididymo-orchitis in 10 of 16 men (62.5%; 95% CI 38.6-81.5), orchitis in 4 (25.0%) and epididymitis in 2 (12.5%). Both women presented with pyelonephritis, one of whom developed a tubo-ovarian abscess requiring drainage. Constitutional features were near-universal (fatigue 100%, night sweats 88.9%, fever 77.8%). Leukocyte counts remained within the reference interval in every patient (mean 5,680/mm3), whereas erythrocyte sedimentation rate (mean 32 mm/h) and C-reactive protein (mean 24.2 mg/L) were only mildly to moderately raised. The Coombs anti-Brucella test was positive in 18 of 18 patients (100%) and the standard agglutination test in 17 of 18 (94.4%), whereas blood culture yielded Brucella melitensis in only 1 of 18 (5.6%). Concomitant extragenitourinary focal disease was present in 8 patients. All patients received doxycycline plus rifampicin; none required modification of the regimen or orchiectomy, and no treatment failure occurred (0 of 18; 95% CI 0-17.6). Median follow-up was 12 weeks (range 6-52) and no relapse was documented within this interval. Conclusions: In endemic regions, genitourinary brucellosis should be suspected in any patient with acute scrotal or flank symptoms accompanied by constitutional complaints, even when the leukocyte count is normal and acute-phase reactants are only marginally elevated. Coombs anti-Brucella serology rather than blood culture is the diagnostic cornerstone, and prompt doxycycline-rifampicin therapy averts surgical intervention.

Metformin is one of the most widely prescribed drugs with a proven safety profile, making it an excellent candidate for drug repurposing strategies. Its anti-Candida effect and its synergistic potential with azoles have already been described against C. albicans and C. glabrata, alongside AMPK involvement and its divergent consequences on autophagy. However, metformin has additional effects on C. albicans biology that remain to be explored using differential proteomics. In the present study, C. albicans SC5314 was treated with metformin to identify the proteins involved in its anti-Candida activity. First, we demonstrated that metformin inhibits C. albicans SC5314 growth in a dose-dependent manner, an effect that intensifies under low glucose (0.2%) and filament-inducing conditions. It also reduces virulence traits such as filamentation, biofilm formation, and invasive growth. Furthermore, it induces significant oxidative stress, which is neutralized by antioxidant agents such as N-acetylcysteine and glutathione. Additionally, 50 mM metformin substantially increased fluconazole efficacy under 0.2% glucose. Our label-free proteomic study of C. albicans SC5314 exposed to 50 mM metformin allowed the identification and quantification of 1,899 proteins, with 95 and 47 proteins showing increased and decreased abundance, respectively. Notably, 26 of the down-regulated proteins were encoded by essential genes, demonstrating the drastic effect of metformin on C. albicans viability. GO Term analysis revealed that the most relevant functions affected were ATP binding, inhibition of ATPase activity (with reduced ATP levels), translation inhibition, mitochondrial function, filamentation, and responses to oxidative stress and antifungals. In conclusion, metformin significantly compromises the viability and virulence of C. albicans at high concentrations. Given that high concentrations of metformin have recently been reported in the intestinal tract, and that intestinal Candida is a known source of invasive infections in immunocompromised, under chemotherapy, and post-surgery patients, understanding metformin anti-Candida action becomes increasingly relevant.

Balamuthia mandrillaris is a free-living amoeba that can cause cutaneous skin lesions, systemic disease, and a severe life-threatening CNS infection, Balamuthia amoebic encephalitis (BAE), which has a high mortality rate (~92%). The current diagnostic methods for B. mandrillaris have many limitations including the long detection time, low sensitivity, and specific diagnostic tests only available in highly specialized centers. The early stage of diagnosis is important for the treatment because of how quickly BAE infection then develops. Therefore, we developed, optimized, and evaluated a novel loop-mediated isothermal amplification (LAMP) assay for the 18S rRNA gene of B. mandrillaris, which can be observed by naked eyes with phenol red. This novel diagnostic assay can detect ten different B. mandrillaris isolates with no cross-reactivity with the DNA of other free-living amoeba, protozoa, fungi, or bacteria tested. Compared with conventional PCR, our LAMP assay displayed ~10-100-fold higher sensitivity from extracted DNA, heat-treated samples, and non-treated samples, with the limit of detection (LOD) of 10 fg/L DNA, 10 trophozoite/100 L for non-treated cells, and 1 trophozoite/100 L for heat-treated cells, respectively. This method was further validated using two newly developed qPCR and dPCR methods to determine the LOD of approximately 1 copy/L. In addition, there is no tolerance for complicated matrices, that LAMP maintained efficacy in cerebrospinal fluid (CSF), urine, and blood-spiked samples. Due to the simplicity, robustness, and high sensitivity of the developed LAMP method, it will be useful for quick clinical detection and diagnosis of B. mandrillaris, particularly in non-specialized centers and resource-limited areas. Here we report the first LAMP diagnostic method for B. mandrillaris; publicly disclosed on Tuesday, November 4th, 2025 at the 20th (XX) International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM 2025) at Puerto Morelos, Mexico.