Journal of medical microbiology最新文献

Introduction. Cryptosporidium presents one of the main waterborne public health threats due to its resistance to chlorine disinfection and ability to cause large-scale outbreaks. The standard method used in the UK water industry for detection and enumeration of Cryptosporidium is based on fluorescence microscopy and is laborious and expensive. Molecular methods such as quantitative polymerase chain reaction (qPCR) can be more amenable to streamlining through automation, improving workflows and standardizing procedures.Hypothesis. The null hypothesis was that there was no difference in the detection or enumeration between the standard method and a qPCR.Aim. We aimed to develop and evaluate a qPCR for the detection and enumeration of Cryptosporidium in drinking water, and to compare the assay with the standard method used in the UK.Methodology. We first developed and evaluated a qPCR method by incorporating an internal amplification control and calibration curve into a real-time PCR currently used for Cryptosporidium genotyping. Then we compared the qPCR assay with the standard method of immunofluorescent microscopy for the detection and enumeration of 10 and 100 Cryptosporidium oocysts in 10 l of artificially contaminated drinking water.Results. The results demonstrated that detection of Cryptosporidium by this qPCR was reliable at low numbers of oocysts; however, enumeration was less reliable and more variable than immunofluorescence microscopy.Conclusions. Despite these results, qPCR offers practical advantages over microscopy. There is potential for the use of PCR-based methods for Cryptosporidium analysis if parts of the upstream sample preparation are revised, and alternative technologies for enumeration (such as digital PCR) are also explored to improve analytical sensitivity.

Introduction. C-type lectin receptors (CLRs) are prominently expressed on myeloid cells where they perform multiple functions including serving as pattern recognition receptors (PRRs) to drive innate as well as adaptive immunity to pathogens. Depending on the presence of a tyrosine-based signalling motif, CLR-microbial pathogen engagement may result in either anti- or pro-inflammatory signalling.Impact statement. In this manuscript, we report our laboratory study of two novel CLRs that recognize Pneumocystis murina cell wall homogenates (CWH) and a purified Pneumocystis carinii cell wall fraction (CWF).Aim. To study the potential of newly generated hFc-CLR fusions on binding to Pneumocystis murina CWHs and P. carinii CWFs and subsequent downstream inflammatory signalling analysis.Methods. Newly generated hFc-CLR fusion CLEC4A and CLEC12B were screened against P. murina CWHs and P. carinii CWFs preparations via modified ELISA. Immunofluorescence assay (IFA) was utilized to visualize hFc-CLR fusion binding against intact fixed fungal life forms to verify results. Quantitative PCR (q-PCR) analysis of lung mRNA from the mouse immunosuppressed Pneumocystis pneumonia (PCP) model versus uninfected mice was employed to detect possible changes in the respective Clec4a and Clec12b transcripts. Lastly, siRNA technology of both CLRs was conducted to determine effects on downstream inflammatory events in mouse macrophages stimulated in the presence of P. carinii CWFs.Results. We determined that both CLEC4A and CLEC12B hFc-CLRs displayed significant binding with P. murina CWHs and P. carinii CWFs. Binding events showed significant binding to both curdlan and laminarin, both polysaccharides containing β-(1,3) glucans as well as N-acetylglucosamine (GlcNAc) residues and modest yet non-significant binding to the negative control carbohydrate dextran. IFA with both CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of both CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that both CLRs were significantly up regulated during infection. Lastly, siRNA of both CLRs in the mouse RAW macrophage cell line was conducted and results demonstrated that silencing of Clec4a resulted in no significant changes in TNF-alpha generation in P. carinii CWF stimulated macrophages. On the contrary, silencing of Clec12b CLR resulted in significant decreases in TNF-alpha in RAW cells stimulated with the same CWF.Conclusion. The data presented here provide new members of the CLRs family recognizing Pneumocystis. Future studies using CLEC4A and/or CLEC12B deficient mice in the PCP mouse model should provide further insights into the host immunological response to Pneumocystis.

Introduction. Pseudomonas aeruginosa in healthcare shower waters presents a high risk of infection to immune-suppressed patients; identifying the colonization-status of water outlets is essential in preventing acquisition.Hypothesis/Gap Statement. Testing frequencies may be insufficient to capture presence/absence of contamination in healthcare waters between sampling and remediation activities. Standardization of outlets may facilitate the management and control of P. aeruginosa.Aim. This study aims to monitor shower waters and drains for P. aeruginosa in augmented and non-augmented healthcare settings every 2 weeks for a period of 7 months during remedial actions.Methodology. All shower facilities were standardized to include antimicrobial silver-impregnated showerhead/hose units, hose-length fixed to 0.8 m and replaced every 3 months. Standard hospital manual decontamination/disinfection occurred daily. Thermostatic-mixer-valves (TMVs) were replaced and disinfected if standard remediation unsuccessful.Results. Of 560 shower and drain samples collected over 14 time-points covering 7 months, P. aeruginosa colonized 40 %(4/10; non-augmented) and 80 %(8/10; augmented-care) showers in the first week. For each week elapsed, new outlets became contaminated with P. aeruginosa by 18-19 % (P<0.001) in shower waters (OR=1.19; CI=1.09-1.31) and drains (OR=1.18; CI=1.09-1.30). P. aeruginosa occurrence in shower water was associated with subsequent colonization of the corresponding drain and vice versa (chi-square; P<0.001) with simultaneous contamination present in 31 %(87/280) of areas. TMV replacement was ineffective in eradicating colonisation in ~83 % of a subset (6/20; three per ward) of contaminated showers.Conclusions. We demonstrate the difficulties in eradicating P. aeruginosa from hospital plumbing, particularly when contamination is no longer sporadic. Non-augmented care settings are reservoirs of P. aeruginosa and should not be overlooked in outbreak investigations. Antimicrobial-impregnated materials may be ineffective once colonization with P. aeruginosa is established beyond the hose and head. Reducing hose-length insufficient to prevent cross-contamination from shower drains. P. aeruginosa colonization can be transient in both drain and shower hose/head. Frequent microbiological monitoring suggests testing frequencies following HTM04-01 guidelines are insufficient to capture the colonization-status of healthcare waters between samples. Disinfection/decontamination is recommended to minimize bioburden and the effect of remediation should be verified with microbiological monitoring. Where standard remediation did not remove P. aeruginosa contamination, intensive monitoring supported justifying replacement of showers and contiguous plumbing.

Introduction. Burkholderia thailandensis is a clinically rare opportunistic pathogen in the genus Burkholderia, and the genomic features and virulence characteristics of B. thailandensis strains that cause human infection remain unclear.Gap Statement. B. thailandensis strains with different virulence induce different host innate immune responses in vitro.Aim. This work aimed to understand the sequence diversity, phylogenetic relationship, and virulence of B. thailandensis BPM causing human infection.Methodology. The comparative molecular and genomic analyses, and mouse infection studies were applied to analyse the virulence and genomic features of B. thailandensis BPM originating from China.Results. The whole genome sequence analysis showed that the genomes of BPM and other avirulent B. thailandensis strains were broadly similar, comprising two highly syntenic chromosomes with comparable numbers of coding regions (CDs), protein family distributions, and horizontally acquired genomic islands. By examining species-specific genomic regions, we obtained molecular explanations for previously known differences in virulence and discovered the potential specific virulence-associated genes of BPM, which likely work together to confer the virulence of BPM. Significantly reduced LD₅₀ and survival rates during mouse infection experiments were found in BPM compared to the avirulent B. thailandensis E264 (BtE264).Conclusion. Taken together, the results of this study provide basic information on the genomic features and virulence characteristics of the virulent B. thailandensis strain BPM, which is helpful for understanding its evolution as it relates to pathogenesis and environmental adaptability.

Introduction. Huangqin Decoction (HQD), a Chinese herbal formula, is widely used for various diseases, including colorectal cancer (CRC).Hypothesis/Gap Statement. We proposed that microbial butyrate mediated PI3K/Akt pathway suppression might involve the anti-cancer effect of HQD.Aim. This study aimed to evaluate the potential mechanism of HQD against CRC.Methodology. An azoxymethane plus dextran sulphate sodium induced CRC mouse model was used, and the intestinal flora and faecal short-chain fatty acid changes were detected, respectively, after HQD administration with 16S rRNA sequencing and gas chromatography coupled with mass spectrometry. Disease activity index, colon length and levels of inflammatory cytokines were measured to evaluate the effect of HQD on intestinal inflammation. Tumour size, number and histopathology were assessed to reflect the impact of HQD on tumour burden. Apoptosis and PI3K/Akt pathway activity were measured by TUNEL staining and Western-blotting. In vitro, the effects of sodium butyrate (NaB) on the viability of CRC cell lines were detected by the Cell-counting Kit-8. The apoptotic cells were determined by TUNEL staining. Cell migration and invasion were assessed by wound healing assay and Transwell assay, respectively. Western-blotting and immunofluorescent staining were used to test the activity of PI3K/Akt pathway.Results. Animal study showed that HQD could improve the gut dysbiosis, increase the abundance of Clostridium and the level of faecal butyric acid. Then, we found that HQD could attenuate colitis, reduce tumour burden, promote cell apoptosis and suppress PI3K/Akt pathway activity in CRC mice. In vitro experiment revealed that NaB treatment could inhibit cell growth, migration and invasion in CRC cell lines. Additionally, NaB enhanced cellular apoptosis, and reduced phosphorylated PI3K and Akt expressions. Interestingly, addition of 740Y-P, an agonist of PI3K, reversed the NaB effects on CRC cells.Conclusion. Overall, in this study, we revealed that HQD could induce apoptosis through microbial butyrate mediated PI3K/Akt inhibition and perform anti-CRC activity.

Introduction. C. difficile infection (CDI) represents an important global threat. In the COVID-19 era, the multifactorial nature of CDI has emerged.Hypothesis - Aim. The aim was to assess the impact of COVID-19 pandemic on the incidence of CDI in a Greek hospital.Methodology. A retrospective study was performed throughout a 51 month period (January 2018 to March 2022), divided into two periods: pre-pandemic (January 2018 to February 2020) and COVID-19 pandemic (March 2020 to March 2022). The effects of the pandemic compared to the pre-pandemic period on the incidence of CDI [expressed as infections per 10 000 bed days (IBD)] were studied using interrupted time-series analysis.Results. Throughout the study, there was an increase in the monthly CDI incidence from 0.00 to 11.77 IBD (P<0.001). Interrupted time-series disclosed an increase in CDI incidence during the pre-pandemic period from 0.00 to 3.36 IBD (P<0.001). During the COVID-19 pandemic period the linear trend for monthly CDI rose from 2.65 to 13.93 IBD (P<0.001). The increase rate was higher during the COVID-19 pandemic period (r₂ = +0.47) compared to the pre-pandemic period (r₁ = +0.16).Conclusion. A significant increase of CDI incidence was observed, with the rate of the rise being more intense during the COVID-19 pandemic.

Introduction. The different pathotypes of Escherichia coli can produce a large number of human diseases. Surveillance is complex since their differentiation is not easy. In particular, the detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 consists of stool culture of a diarrhoeal sample on enriched and/or selective media and identification of presumptive colonies and confirmation, which require a certain level of training and are time-consuming and expensive.Hypothesis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a quick and easy way to obtain the protein spectrum of a microorganism, identify the genus and species, and detect potential biomarker peaks of certain characteristics.Aim. To verify the usefulness of MALDI-TOF MS to rapidly identify and differentiate STEC O157 : H7 from other E. coli pathotypes.Methodology. The direct method was employed, and the information obtained using Microflex LT platform-based analysis from 60 clinical isolates (training set) was used to detect differences between the peptide fingerprints of STEC O157 : H7 and other E. coli strains. The protein profiles detected laid the foundations for the development and evaluation of machine learning predictive models in this study.Results. The detection of potential biomarkers in combination with machine learning predictive models in a new set of 142 samples, called 'test set', achieved 99.3 % (141/142) correct classification, allowing us to distinguish between the isolates of STEC O157 : H7 and the other E. coli group. Great similarity was also observed with respect to this last group and the Shigella species when applying the potential biomarkers algorithm, allowing differentiation from STEC O157 : H7Conclusion. Given that STEC O157 : H7 is the main causal agent of haemolytic uremic syndrome, and based on the performance values obtained in the present study (sensitivity=98.5 % and specificity=100.0 %), the implementation of this technique provides a proof of principle for MALDI-TOF MS and machine learning to identify biomarkers to rapidly screen or confirm STEC O157 : H7 versus other diarrhoeagenic E. coli in the future.

Introduction. Bosnia and Herzegovina (B and H) has been recognized for decades as a country with a high risk of diseases caused by hantaviruses.Gap statement. The severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) pandemic has diverted attention from many pathogens, including hantavirus.Aim. To provide a socio-demographic, temporal, geographical and clinical laboratory overview of the expansion of hantavirus infection cases during the SARS-CoV-2 pandemic in B and H in 2021.Methodology. The RecomLine HantaPlus IgG, IgM immuno-line assay (Mikrogen, Germany) was used to detect IgG and IgM antibodies to hantavirus serotypes in human sera from clinically suspected cases.Results. In 2021 (January-October), the number of confirmed cases of hantavirus infection and tested persons (92/140; 65,71 %) was higher than in the previous 2 years, 2020 (2/20; 10.00 %) and 2019 (10/61; 16.39 %). Most of the infected persons were men (84/92; 91.30 %). Hantavirus infections were recorded from January to October 2021, and the peak was reached in July (25/92; 27.17 %). Six out of 10 cantons in the Federation of Bosnia and Herzegovina (FB and H) were affected, namely Sarajevo Canton, Central Bosnia Canton, Neretva Canton, Zenica-Doboj Canton, Posavina Canton and Bosnian-Podrinje Canton Goražde, in descending order. Of the 38/92 (41.30 %) infected patients with characteristic clinical manifestations of haemorrhagic fever, including renal (mainly) or pulmonary syndrome, 32/92 (34.78 %) were hospitalized in the Clinical Center of the University of Sarajevo. Two cases were detected with dual infection, hantavirus (Puumala) with Leptospira in one and SARS-CoV-2 in another case. The largest number of infections was related to Puumala (PUUV) (83/92; 90.22 %), while the rest of the infections were caused by the hantavirus Dobrava serotype (DOBV).Conclusion. The reported infections were probably caused by exposure of individuals to at-risk areas inhabited by contaminated rodents as natural reservoirs of hantavirus. As a highly endemic area, B and H requires continuous monitoring and increased awareness of this problem.

Recurrent vulvovaginal candidiasis (RVVC) is a microbial, immune and sexual health disorder impacting up to 10 % of the adult female population. Fluconazole is a well-established antifungal drug commonly utilized for acute and long-term RVVC treatment. This insight review provides an overview of known vaginal and gastrointestinal microbiota characteristics in RVVC, presents the potential impacts of fluconazole therapy on multi-microbiome relationships and discusses implications for future research and clinical practice. Next-generation sequencing (NGS) and molecular methods to accurately define vaginal microbiota trends in RVVC are not comprehensively available, limiting understanding of microbiota roles in RVVC. Inconsistencies and variances in Lactobacillus profiles in RVVC women suggest poorly understood disease implications on the bacterial and fungal microbiomes. Investigations of environmental conditions like vaginal pH, drug therapy's impact, especially fluconazole maintenance therapy, and the elucidation of multi-microbiome relationships in RVVC are required to further investigate disease pathogenesis and responsible antimicrobial prescribing.