- Volume 72, Issue 5, 2023

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.

Introduction.   Klebsiella pneumoniae   is a major threat to public health worldwide. It is the causative agent for multiple disease presentations including urinary tract infection, septicemia, liver abscess, wound infection and respiratory tract infection.   K. pneumoniae   causes community- and hospital-acquired pneumonia, which is a devastating disease associated with high mortality rates.

Hypothesis. There is a growing concern about the emergence of multidrug-resistant   K. pneumoniae   strains complicating the treatment with the current available therapeutics; therefore, there is an urgent need for the development of new antimicrobial agents.

Aim.   K. pneumoniae   causes an acute respiratory disease in mice and in the current work we investigated the capability to perform non-invasive monitoring of bioluminescent   Klebsiella   to monitor therapeutic efficacy.

Methodology. We engineered a bioluminescence reporter strain of   K. pneumoniae   to monitor the impact of antibiotics in a murine respiratory disease model.

Results. We demonstrate that bioluminescence correlates with bacterial numbers in host tissues allowing for a non-invasive enumeration of bacterial replication in vivo. Light production is directly linked to bacterial viability, and this novel bioluminescent   K. pneumoniae   strain enabled monitoring of the efficacy of meropenem therapy in arresting bacterial proliferation in the lung.

Conclusion. The use of non-invasive bioluminescent imaging improves preclinical animal model testing to detect study outcome earlier and with higher sensitivity.

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. 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 : H7

Conclusion. 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.

Introduction. Azelastine hydrochloride, a second-generation histamine H1 receptor (H1R) antagonist, exhibits anti-chlamydial effects against Chlamydia trachomatis (CT) in HeLa cells (genital infection model).

Hypothesis/Gap Statement. Non-antibiotic pharmaceutical interactions with CT are an understudied field and the anti-chlamydial effects of azelastine are a potential interaction requiring further elucidation.

Aim. To explore the underlying anti-chlamydial mechanisms of azelastine.

Methodology. We assessed the specificity of azelastine for the chlamydial species and host cell type, the timing of azelastine application and whether the anti-chlamydial effects could be reproduced with different H1R-modulating compounds.

Results. We observed similar anti-chlamydial azelastine effects for Chlamydia muridarum as well as for an ocular CT strain in human conjunctival epithelial cells (ocular infection model). Pre-incubating host cells with azelastine before infection mildly reduced chlamydial inclusion numbers and infectivity. Incubation of cells with azelastine initiated concomitantly with the chlamydial infection, or initiated several hours post-infection, reduced inclusion size, number and infectivity, and altered chlamydial morphology. These effects were strongest when azelastine was added shortly after or with the infection. Azelastine effects were not alleviated by increased concentrations of culture medium nutrients. Additionally, we did not observe anti-chlamydial effects when incubating cultures either with a different H1R antagonist or agonist, indicating that azelastine effects are probably H1R-independent.

Conclusion. Accordingly, we conclude that azelastine anti-chlamydial effects are not restricted to a specific chlamydial species, strain or culture model, and are probably not mediated by H1R antagonism. Thus, it appears likely that off-target mechanisms of azelastine may explain our observations.

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.