Journal of medical microbiology最新文献

Introduction . Cryptococcus neoformans and Mycobacterium tuberculosis (MTb) are opportunistic pathogens that share overlapping geographical distributions and physiological niches within the human body. Both are recognized by the World Health Organization as high-priority pathogens.Gap Statement. Although clinical reports of co-infections with cryptococcosis and tuberculosis are increasing, experimental studies exploring their interactions remain scarce.Aim. We aimed to observationally evaluate whether C. neoformans isolates would alter morphology when co-cultured with Mycobacteria spp. and observe how these changes might alter the host immune response to C. neoformans cells.Methodology. We cultured C. neoformans reference strain and clinical isolates in physiologically relevant growth media, in the presence or absence of Mycobacterium spp. Then, we generated alveolar-like macrophages and created a stimulation environment similar to a tuberculosis environment to perform phagocytic killing assays of C. neoformans cells.Results. Here, we demonstrate that C. neoformans can grow in the presence of either heat-killed MTb antigen or the live vaccine strain, Mycobacterium bovis BCG. In response to the presence of mycobacteria, C. neoformans increased in number and exhibited enhanced virulence-associated traits, including titan cell formation, capsule enlargement and increased survival from phagocytosis.Conclusion. This work provides proof of principle for a dynamic, inter-pathogen interaction that may contribute to the exacerbation of disease outcomes in settings of a co-infection.

Introduction. Direct detection of Borrelia burgdorferi by culture is considered the gold standard for confirming Lyme disease (LD). However, B. burgdorferi culture is not routinely used in clinical practice or research due to its lengthy protocol and low success rate. This study aimed to streamline the process by integrating a specific quantitative PCR (qPCR) screening early into the B. burgdorferi culture workflow for identification of cultures that are likely to yield viable spirochetes.Methods. Thirty-two blood plasma and 11 cerebrospinal fluid (CSF) samples were collected from 32 children with serologically confirmed LD and incubated in modified Kelly-Pettenkofer medium for up to 9 weeks, with weekly assessments for viable spirochetes using microscopy. After 3 weeks, the presence of B. burgdorferi DNA in culture was assessed by qPCR targeting the B. burgdorferi flagellin B gene. The estimated copy number of the target template was compared to the assay's 95% limit of detection (LOD).Results. After 9 weeks of incubation, viable spirochetes were observed in 2 (n=2/32, 6.3%) plasma cultures and 3 (n=3/11, 27.3%) CSF cultures. These were only observed in cultures showing copy numbers above 95% LOD in qPCR testing at week 3 (n=2/3 plasma cultures, 66.7%; n=3/3 CSF cultures, 100.0%).Conclusion. Culturing B. burgdorferi is challenging and, despite a high workload, often not successful. qPCR may serve as an effective screening tool for B. burgdorferi cultures, enabling the culturing process to be streamlined by prioritizing cultures with target copy numbers exceeding the 95% LOD of the qPCR assay.

Introduction. Recently, flow cytometry has gained the attention of clinical microbiologists for its ability to characterize bacterial species. This article shows how acoustic-enhanced flow cytometry, combined with the fluorescent dye SYTO 9, can differentiate between Gram-positive and Gram-negative bacteria.Gap Statement. SYTO 9 is a cell membrane-permeable dye with a high affinity for nucleic acids in both living and non-living prokaryotic and eukaryotic cells and has been used as a counterstain to discriminate between live and dead cells in combination with other dyes. However, the consistency of its cell permeability in different bacterial species and its potential application to Gram differentiation has not been fully considered.Aim. We sought to assess the suitability of the fluorescent dye, SYTO 9, to differentiate Gram-positive and Gram-negative bacteria by flow cytometry.Methodology. A range of common Gram-positive and Gram-negative bacterial species were stained with SYTO 9, then processed using an acoustic-enhanced flow cytometer (Attune NxT, Thermo Fisher). The fluorescence emission data were gated and analysed in quadrant plots.Results. Single and polymicrobial bacterial suspensions stained with SYTO 9 produced different fluorescence signals in Gram-positive and Gram-negative bacteria in the forward scatter-height/blue 3-height (FSC-H/BL3-H) quadrant. Gram-positive species (Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus pneumoniae and Streptococcus pyogenes) had higher fluorescence intensities in the BL3 channel than the Gram-negative species (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia thailandensis and Proteus vulgaris) in both single and mixed cultures.Conclusion. The FSC-H/BL3-H quadrant analysis of flow cytometer emission spectra from SYTO 9-stained bacterial suspensions segregated Gram-positive and Gram-negative bacteria into separate quadrants based on their different fluorescence intensities. This provides a single-dye flow cytometry method for Gram differentiation.

Introduction. Bacterial infections of skin wounds can increase hospitalization duration and lead to worse patient prognoses, especially for burn wounds and diabetic foot ulcers. The two main pathogens which infect these wounds are Pseudomonas aeruginosa and Staphylococcus aureus. However, many other species can be present in wound infections, including skin commensal bacteria such as Staphylococcus epidermidis and Micrococcus luteus.Hypothesis. It was hypothesized that co-infection alters the antibiotic resistance of each species present.Aim. To investigate dual-species commensal-pathogen co-culture and assess the potential influence on the antibiotic resistance of each species.Methodology. The commensal and pathogenic species were grown either separately or in dual-species co-culture, potentially allowing biofilm formation for 24 h and were subsequently treated with antibiotics (ciprofloxacin or tobramycin). The impact of the co-culture growth was compared with single species cultures and the effect of the antimicrobial treatment on both conditions were assessed through Minimum Biofilm Eradication Concentrations (MBECs) and bacterial viable counts.Results. The viability of each bacterial species was reduced in the presence of other species, and this translated to reduced antibiotic resistance (lower MBECs) of P. aeruginosa in particular. The resistance of the other species appeared more dependent on the specific inter-species effects.Conclusion. The inclusion of a commensal species with pathogens in co-culture reduced the antibiotic resistance, and inter-species effects influenced the viability of the pathogens. More realistic antimicrobial resistance assessment protocols accounting for microbial communities could therefore lead to more effective treatments.

Candida glabrata is an opportunistic fungal pathogen and a leading cause of invasive candidiasis, particularly in immunocompromised patients, where treatment is increasingly compromised by intrinsic and acquired antifungal resistance. Despite lacking morphological plasticity, C. glabrata employs distinct virulence strategies, including adhesin-mediated host colonization, intracellular survival within phagocytes, stress tolerance, iron acquisition and biofilm formation. This review synthesizes current knowledge of C. glabrata virulence and antifungal resistance mechanisms, with a particular focus on azole and echinocandin resistance driven by efflux pump regulation and FKS mutations. We critically evaluate the greater wax moth larva, Galleria mellonella, as a non-mammalian in vivo model for studying C. glabrata pathogenesis, host-pathogen interactions and antifungal efficacy. Evidence demonstrating concordance between G. mellonella, murine models and clinical outcomes is discussed, alongside the model's limitations, including the absence of adaptive immunity. Collectively, this review highlights G. mellonella as a robust, cost-effective platform for dissecting C. glabrata virulence traits and for preclinical screening of antifungal therapies, supporting its growing role in translational fungal research.

Introduction. The increasing resistance and the pathogen's complex multi-drug resistance mechanisms made the selection of effective antimicrobial treatments more challenging for Pseudomonas aeruginosa (P. aeruginosa). The study aimed to explore the effect of Scutellaria baicalensis, Prunella vulgaris and antimicrobial peptide LL-37 on the virulence factors of P. aeruginosa.Hypothesis. Previous studies have shown that extracts from traditional Chinese medicines, Scutellaria baicalensis and Prunella vulgaris, can also enhance the effects of antibiotics and reduce antibiotic resistance in P. aeruginosa. Antimicrobial peptide LL-37 shows the potential as a new-generation candidate for treating multi-drug-resistant bacteria, which has advantages over traditional antibiotics, whilst the combination role between Scutellaria baicalensis, Prunella vulgaris and LL-37 in P. aeruginosa remains unknown.Aim. We explored whether the combined use of Scutellaria baicalensis, Prunella vulgaris and LL-37 can exert antibacterial effects through the quorum sensing (QS) system.Methodology. The minimal inhibitory concentrations of Scutellaria baicalensis, Prunella vulgaris and LL-37 were determined for PAO1 and PA-ΔlasI/rhlI using micro broth dilution. The antibacterial activity of Scutellaria baicalensis combined with LL-37 and Prunella vulgaris combined with LL-37 was also assessed. The growth abilities of PAO1 were analysed after being treated with Scutellaria baicalensis, Prunella vulgaris and LL-37, respectively. Elastase secretion was measured using Congo red-elastic proteinase assays. And the expressions of QS genes (lasI, rhlR) were analysed by real-time PCR.Results. Single or combined treatments of Scutellaria baicalensis and LL-37 and Prunella vulgaris and LL-37 would significantly reduce elastase secretion. There were no significant differences in proliferation between the groups at any timepoint. All treatments downregulated lasI and rhlR gene expressions.Conclusion. Scutellaria baicalensis, Prunella vulgaris and antimicrobial peptide LL-37 all down-regulate the QS system-related genes of P. aeruginosa, inhibiting the secretion of virulence factors and reducing bacterial toxicity.

Dientamoeba fragilis is a gastrointestinal parasite of controversial clinical significance. From its discovery until today, contradictory articles have been published on whether infection is correlated with symptoms, treatment is associated with recovery and whether infection is associated with elevated intestinal inflammatory markers (faecal calprotectin). Additionally, there is no consensus on the infective stage of the lifecycle. Competing theories propose that either Enterobius vermicularis ova act as a vector for the transmission of trophozoites or that the cyst stage, which is rarely found, is responsible for infection. In this review, we aim to critique these contradictions to determine if D. fragilis should be considered a pathogen in clinical practice. The frequent limitation of studies is challenges in setting up a reliable, healthy control group and the reliability of diagnostic methods. Many studies are opportunistic in design, using samples that have been submitted for routine pathology testing. Even if all pathology tests are negative for infectious agents, the current health status of people who are submitting samples for pathology testing is unlikely to be the best option, just the most available one. Of greater concern is the reliability of some diagnostic methods. Some studies have suggested that at least one of the lab-based real-time PCR assays used for the diagnosis of D. fragilis has issues with false positives in human samples. This calls into question much of the evidence that has been published on D. fragilis being a commensal instead of a pathogen. As such, D. fragilis should be considered a potential pathogen when investigating gastrointestinal illness. Developing better guidelines on determining when D. fragilis is the causative agent of symptoms and when to treat are important topics for future research.

Introduction. Chronic wounds are notoriously difficult to treat and are associated with decreased limb function, reduced quality of life and significant morbidity. Their recurrent nature, despite aggressive antibiotic therapy, is due in part to the presence of polymicrobial biofilms. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most frequently co-isolated pathogens in these infections and are known to form complex biofilms that hinder treatment.Hypothesis. We hypothesized that co-existence and competitive dynamics between P. aeruginosa and S. aureus in chronic wound infections are influenced by strain-specific interactions and may not rely solely on well-characterized inhibitory mechanisms such as 2-alkyl-4-quinolone (AQ) production by P. aeruginosa impacting on S. aureus fitness.Aim. To establish a polymicrobial chronic wound infection model and assess the contribution of AQ signalling and strain-specific interactions on co-existence.Methodology. We used a modified chronic wound biofilm model to co-culture matched and mismatched clinical isolate pairs of P. aeruginosa and S. aureus, collected from two different chronic wound patients. Viable bacterial counts (c.f.u.) were quantified over an 8-day period. AQ production by each P. aeruginosa strain was quantified using liquid chromatography-MS.Results. A stable culture of P. aeruginosa strains was achieved, but distinct behaviours between each S. aureus strain were seen. One matched clinical isolate pair maintained stable c.f.u. levels of both species throughout the 8-day model, indicating a compatible co-existence. In contrast, mismatched pairs showed early loss of S. aureus viability and the emergence of small colony variants after 4 days, not seen in matched pair growth. Interestingly, the most competitive P. aeruginosa strain exhibited undetectable levels of all AQs tested, indicating that its dominance was not due to AQ-mediated antagonism, as has previously been described.Conclusion. Our findings demonstrate that stable dual-species biofilm formation in chronic wounds is strain-dependent and that P. aeruginosa can impact on S. aureus fitness through AQ-independent mechanisms. These results highlight the importance of using clinical isolates in biofilm research and caution against generalizing findings from laboratory strains to complex clinical infections.

Introduction. Growing evidence indicates significant interactions between the intestinal microflora and drugs commonly used to treat coronary heart disease.Hypothesis/Gap Statement. Despite this, research specifically investigating the relationship between proprotein convertase subtilisin/kexin type 9 inhibitors and alterations in the gut microbiota has not been previously published.Aim. This study aimed to identify changes in the gut microbiota potentially associated with evolocumab use in patients with acute myocardial infarction (AMI).Methodology. In this prospective study, 26 AMI patients receiving statins (≥8 weeks) were administered evolocumab (420 mg/4 weeks) alongside standard therapy. Eighteen age-matched healthy volunteers served as controls. 16S rRNA sequencing (NCBI SRA: PRJNA1154993) was subsequently performed on samples from these groups to analyse the gut microbiota community.Results. No significant α-diversity differences were observed among groups (P>0.05). Firmicutes dominated AMI-evolocumab baseline (0 W: 76.32%) versus post-treatment (8 W: 65.22%) and controls (60.40%), while Bacteroidota increased post-treatment (0 W: 15.07%→8 W: 19.99%; control: 27.11%). The second most abundant phyla were Proteobacteria and Actinobacteria. In addition, the differences in the microbial structure among the three groups were as follows: at the genus level, the results of the genus difference analysis revealed significant differences in the abundances of nine types of bacteria among the three groups. Compared with those in the AMI-evolocumab (0 W) group, the abundances of beneficial bacteria, such as Odoribacter and Parabacteroides, were increased in the AMI-evolocumab (8 W) group.Conclusion. Our research showed that evolocumab can regulate the gut microbiota of patients with AMI to promote a healthier state, which is beneficial for patients with AMI.

Introduction. The correlation between antibiotic exposure and adverse outcomes in patients with acute-on-chronic liver failure (ACLF) remains controversial, and the underlying mechanism is unclear.Hypothesis/Gap Statement. This study hypothesizes that antibiotic exposure in ACLF patients alters gut microbiota, which affects the outcome of ACLF.Aim. To explore the effect of antibiotic exposure on gut microbiota that affects the outcome of ACLF.Methodology. A retrospective matched study of ACLF patients and the ACLF rat model was used to assess adverse outcomes associated with antibiotic exposure. The gut microbiota of the ACLF patients and the ACLF rat model were sequenced using the Illumina MiSeq platform.Results. Twenty-three ACLF patients who were exposed to antibiotics and 46 matched controls who were not exposed to antibiotics were enrolled. The survival rates at 4, 12 and 24 weeks were significantly lower in the exposure group than in the non-exposure group. In the ACLF rat model, hepatitis in the antibiotic-exposure group became more severe, and the alanine transaminase levels were higher than those of the non-exposure group. The gut microbiota diversity was decreased in the ACLF patients with antibiotic exposure, and the proportions of Enterococcaceae and Peptostreptococcaceae were increased, while those of Lachnospiraceae, Bifidobacteriaceae and Bacteroidaceae were decreased. In the rat model, antibiotic exposure induced Gram-positive and Gram-negative bacterial eradication, and Klebsiella became the dominant micro-organism.Conclusion. Antibiotic exposure aggravated hepatitis and had no survival benefit for ACLF. The underlying mechanism may be related to dysbiosis in the gut microbiota.