A possible mechanism of farnesol tolerance in C. albicans biofilms implemented by activating the PKC signalling pathway and stabilizing ROS levels

Introduction. Biofilms are the natural growth state for most microorganisms. C. albicans biofilms are composed of multiple cell types (round budding yeast-form cells, oval pseudohyphal cells, and elongated hyphal cells) encased in an extracellular matrix. C. albicans biofilms are notorious for resistance to antimicrobial treatments, a property that might be determined by complex mechanisms. Exogenous farnesol exerts a certain antifungal activity against C. albicans with medical implications. Different from other microbes, C. albicans biofilms can tolerate exogenous farnesol at high concentration with some cells still surviving and even maintaining proliferation, but the mechanism is unclear. Hypothesis. The study hypothesizes that C. albicans resists farnesol by activating the PKC signalling pathway. Aim. The study aims to discuss the molecular mechanism of C. albicans resistance to farnesol. Methodology. The ROS levels, the genes and proteins of the PKC pathway were compared between the farnesol-tolerant and non-tolerant groups using ROS levels assay, q-RT PCR and Western blot, respectively. Further, the mutant strains (pkc1Δ/Δ and mkc1Δ/Δ) were constructed, then the survival rates and ROS levels of biofilms exposed to farnesol were compared between mutant and wild strains. The morphological changes were observed using TEM. Results. The survival rates of C. albicans biofilms decreased rapidly under the lower concentration of farnesol (P<0.05), and kept stable (P>0.05) as the concentration rose up to 200 µM. The gene expression of the PKC pathway increased, while ROS levels remained stable and even decreased in the farnesol-tolerant biofilms, compared with those in the farnesol-nontolerant biofilms after farnesol treatment (P<0.05); pkc1 and mkc1 were significantly upregulated by C. albicans during the development of biofilm tolerance to farnesol. The cell wall and cytoplasm of pkc1Δ/Δ and mkc1Δ/Δ were damaged, and the ROS level increased (P<0.05); meanwhile, the survival rate of biofilms decreased compared with that of wild-type strain under the same farnesol concentrations (P<0.05). ROS inhibitors reversed these changes in pkc1Δ/Δ and mkc1Δ/Δ when the mutant strains exposed to farnesol. Conclusion. C. albicans biofilms can tolerate high concentrations of farnesol by activating pkc1 and mkc1 of the PKC pathway and stabilizing ROS levels. The pkc1 and mkc1 are two key genes regulated by C. albicans in the process of biofilm tolerance to farnesol.

Environment in the lung of cystic fibrosis patients stimulates the expression of biofilm phenotype in Mycobacterium abscessus

Introduction. Pulmonary infections caused by organisms of the Mycobacterium abscessus complex are increasingly prevalent in populations at risk, such as patients with cystic fibrosis, bronchiectasis and emphysema. Hypothesis. M. abscessus infection of the lung is not observed in immunocompetent individuals, which raises the possibility that the compromised lung environment is a suitable niche for the pathogen to thrive in due to the overproduction of mucus and high amounts of host cell lysis. Aim. Evaluate the ability of M. abscessus to form biofilm and grow utilizing in vitro conditions as seen in immunocompromised lungs of patients. Methodology. We compared biofilm formation and protein composition in the presence and absence of synthetic cystic fibrosis medium (SCFM) and evaluated the bacterial growth when exposed to human DNA. Results. M. abscessus is capable of forming biofilm in SCFM. By eliminating single components found in the medium, it became clear that magnesium works as a signal for the biofilm formation, and chelation of the divalent cations resulted in the suppression of biofilm formation. Investigation of the specific proteins expressed in the presence of SCFM and in the presence of SCFM lacking magnesium revealed many different proteins between the conditions. M. abscessus also exhibited growth in SCFM and in the presence of host cell DNA, although the mechanism of DNA utilization remains unclear. Conclusions. In vitro conditions mimicking the airways of patients with cystic fibrosis appear to facilitate M. abscessus establishment of infection, and elimination of magnesium from the environment may affect the ability of the pathogen to establish infection.

The human bile salt sodium deoxycholate induces metabolic and cell envelope changes in Salmonella Typhi leading to bile resistance

Introduction. Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of typhoid fever. To establish an infection in the human host, this pathogen must survive the presence of bile salts in the gut and gallbladder. Hypothesis. S. Typhi uses multiple genetic elements to resist the presence of human bile. Aims. To determine the genetic elements that S. Typhi utilizes to tolerate the human bile salt sodium deoxycholate. Methodology. A collection of S. Typhi mutant strains was evaluated for their ability to growth in the presence of sodium deoxycholate and ox-bile. Additionally, transcriptomic and proteomic responses elicited by sodium deoxycholate on S. Typhi cultures were also analysed. Results. Multiple transcriptional factors and some of their dependent genes involved in central metabolism, as well as in cell envelope, are required for deoxycholate resistance. Conclusion. These findings suggest that metabolic adaptation to bile is focused on enhancing energy production to sustain synthesis of cell envelope components exposed to damage by bile salts.

Comparative analyses of clinical features reveal the severity of human adenovirus type 55 and type 7 in acute respiratory tract infections

Introduction. Human adenovirus (HAdV) is an important pathogen in acute respiratory tract infections (ARTIs) and HAdV genotypes are associated with disease severity. Hypothesis. Comparative analyses of clinical features could reveal the severity of different HAdV genotypes in ARTIs. Aim. This study aimed to investigate the molecular epidemiology of HAdV infections and explore the correlations between clinical features and HAdV genotypes. Methodology. A retrospective study was conducted on ARTIs at Beijing Chao-Yang Hospital during the period 2011–2016. A standardized data form was used to record the clinical information. HAdV was detected by FQ-PCR from respiratory specimens, and genotypes were determined by entire hexon gene sequencing. Results. A total of 8044 samples were collected, of which 296 (3.7 %) were HAdV-positive. Patients ≤44 years old were more likely to be positive for HAdV. There were three peak periods of adenoviral infections, with detection rates of 13.03, 9.39 and 10.38 %, respectively. Six HAdV genotypes (HAdV-55, -7, -3, -14, -50, -2) were identified, with HAdV-55 and HAdV-7 being the most prevalent (50.6 and 21.5 %). Compared with HAdV-7 and other types, patients infected with HAdV-55 had a longer duration of fever (P=0.0428). Infections with HAdV-55 and HAdV-7 were more severe compared to those caused by other types, with higher rates of oxygen therapy and mechanical ventilation (P=0.0172 and P=0.0144). All five deaths were caused by HAdV‐55. Conclusion. This study describes the epidemiological characteristics of HAdV infections in North China, revealing the higher severity of HAdV-55 and HAdV-7 in ARTIs. Thus, strengthened surveillance of HAdV genotypes is warranted.

VicRK and CovR polymorphisms in Streptococcus mutans strains associated with cardiovascular infections

Introduction. Streptococcus mutans , a common species of the oral microbiome, expresses virulence genes promoting cariogenic dental biofilms, persistence in the bloodstream and cardiovascular infections. Gap statement. Virulence gene expression is variable among S. mutans strains and controlled by the transcription regulatory systems VicRK and CovR. Aim. This study investigates polymorphisms in the vicRK and covR loci in S. mutans strains isolated from the oral cavity or from the bloodstream, which were shown to differ in expression of covR, vicRK and downstream genes. Methodology. The transcriptional activities of covR, vicR and vicK were compared by RT-qPCR between blood and oral strains after exposure to human serum. PCR-amplified promoter and/or coding regions of covR and vicRK of 18 strains (11 oral and 7 blood) were sequenced and compared to the reference strain UA159. Results. Serum exposure significantly reduced covR and vicR/K transcript levels in most strains (P<0.05), but reductions were higher in oral than in blood strains. Single-nucleotide polymorphisms (SNPs) were detected in covR regulatory and coding regions, but SNPs affecting the CovR effector domain were only present in two blood strains. Although vicR was highly conserved, vicK showed several SNPs, and SNPs affecting VicK regions important for autokinase activity were found in three blood strains. Conclusions. This study reveals transcriptional and structural diversity in covR and vicR/K, and identifies polymorphisms of functional relevance in blood strains, indicating that covR and vicRK might be important loci for S. mutans adaptation to host selective pressures associated with virulence diversity.

National trends in hospital, long-term care and outpatient Acinetobacter baumannii resistance rates

Introduction. Acinetobacter baumannii is a top-priority pathogen of the World Health Organization (WHO) and the Centers for Disease Control (CDC) due to antibiotic resistance. Gap Statement. Trends in A. baumannii resistance rates that include community isolates are unknown. Aim. Identify trends in A. baumannii resistance rates across the Veterans Affairs (VA) Healthcare System, including isolates from patients treated in hospitals, long-term care facilities and outpatient clinics nationally. Methodology. We included A. baumannii clinical cultures collected from VA patients from 2010 to 2018. Cultures were categorized by location: VA medical centers (VAMCs), long-term care (LTC) units [community living centers (CLCs)], or outpatient. We assessed carbapenem resistance, multidrug resistance (MDR) and extensive drug resistance (XDR). Time trends were assessed with Joinpoint regression. Results. We identified 19 376 A . baumannii cultures (53% VAMCs, 4% CLCs, 43% outpatient). Respiratory cultures were the most common source of carbapenem-resistant (43 %), multidrug-resistant (49 %) and extensively drug-resistant (21 %) isolates. Over the study period, the number of A. baumannii cultures decreased significantly in VAMCs (11.9% per year). In 2018, carbapenem resistance was seen in 28% of VAMC isolates and 36% of CLC isolates, but only 6% of outpatient isolates, while MDR was found in 31% of VAMC isolates and 36% of CLC isolates, but only 8 % of outpatient isolates. Carbapenem-resistant, multidrug-resistant and extensively drug-resistant A. baumannii isolates decreased significantly in VAMCs and outpatient clinics over time (VAMCs: by 4.9, 7.2 and 6.9%; outpatient: by 11.3, 10.5 and 10.2% per year). Resistant phenotypes remained stable in CLCs. Conclusion. In the VA nationally, the prevalence of A. baumannii is decreasing, as is resistance. Carbapenem-resistant and multidrug-resistant A. baumannii remain common in VAMCs and CLCs. The focus of infection control and antimicrobial stewardship efforts to prevent transmission of resistant A. baumannii should be in hospital and LTC settings.

Use of Sysmex UF-5000 flow cytometry in rapid diagnosis of urinary tract infection and the importance of validating carryover rates against bacterial count cut-off

Introduction. Urinary tract infections are common bacterial infections worldwide. Urine culture is the gold standard method to identify and quantify the presence or absence of bacteria in urine. Flow cytometry, which can differentiate and quantify multiple particles (including bacteria) in the urine, presents an alternative method for rapid screening to rule out bacteriuria. Hypothesis. Adding flow cytometry to identify urine samples without bacteriuria could substantially reduce the number of urine samples that need to be cultured as well as the response time for negative results. However, the level of instrument rinsing between samples could affect sample-to-sample carryover rate, a concept given little attention in previous studies. Aim. We aimed to evaluate urine flow cytometry as a rapid screening method to identify urine samples without significant bacterial growth, including analyses of cross-contamination and sample-to-sample carryover rate. Methodology. We analysed 3919 urine samples by quantitative urine culture and flow cytometry screening (Sysmex UF-5000). Receiver operator characteristic (ROC) curve analyses were used to test method agreement to identify: (a) positive vs. negative culture and (b) mixed vs. pure culture. In addition, we performed carryover and cross-contamination studies. Results. ROC curve analyses identified bacterial count (BACT ml−1) and leucocyte count (WBC µl−1) as possible predictors of bacterial growth in the total material and subpopulations, except pregnant women (n=451). This subgroup was excluded from further analyses, leaving a final 3468 urine samples. Area under the ROC curve was 0.94 (95 % CI 0.93–0.95) and 0.81 (95 % CI 0.79–0.82) for bacterial and leucocyte count, respectively. A bacterial count cut-off of 30 BACT ml−1 resulted in 95.2 % sensitivity and 91.2 % negative predictive value, resulting in approximately 30 % of urine samples that could be reported as negative without culture. Use of high-level rinse modes was necessary to ensure carryover rates <0.05 %. Conclusion. Flow cytometry is a suitable and rapid method to rule out urine samples without significant bacterial growth. Rinses between samples should be adjusted, depending on the cut-off used, to prevent sample-to-sample carryover, whereas cross-contamination can be eliminated by the use of separate urine aliquots for flow cytometry analysis and urine culturing respectively.

Treating autism spectrum disorder by intervening with gut microbiota

Autism spectrum disorder (ASD) comprises a group of neurodevelopmental disorders with a high prevalence in childhood. The gut microbiota can affect human cognition and moods and has a strong correlation with ASD. Microbiota transplantation, including faecal microbiota transplantation (FMT), probiotics, breastfeeding, formula feeding, gluten-free and casein-free (GFCF) diet and ketogenic diet therapy, may provide satisfying effects for ASD and its related various symptoms. For instance, FMT can improve the core symptoms of ASD and gastrointestinal symptoms. Probiotics, breastfeeding and formula feeding, and GFCF diet can improve gastrointestinal symptoms. The core symptom score still needs to be confirmed by large-scale clinical randomized controlled studies. It is recommended to use a ketogenic diet to treat patients with epilepsy in ASD. At present, the unresolved problems include which of gut the microbiota are beneficial, which of the microorganisms are harmful, how to safely and effectively implant beneficial bacteria into the human body, and how to extract and eliminate harmful microorganisms before transplantation. In future studies, large sample and randomized controlled clinical studies are needed to confirm the mechanism of intestinal microorganisms in the treatment of ASD and the method of microbial transplantation.

Comparing hospital-resource utilization by an enhanced pneumonia surveillance programme for COVID-19 with pre-pandemic pneumonia admissions – a Singaporean hospital’s experience

Introduction. During the early days of coronavirus disease 2019 (COVID-19) in Singapore, Tan Tock Seng Hospital implemented an enhanced pneumonia surveillance (EPS) programme enrolling all patients who were admitted from the Emergency Department (ED) with a diagnosis of pneumonia but not meeting the prevalent COVID-19 suspect case definition. Hypothesis/Gap Statement. There is a paucity of data supporting the implementation of such a programme. Aims. To compare and contrast our hospital-resource utilization of an EPS programme for COVID-19 infection detection with a suitable comparison group. Methodology. We enrolled all patients admitted under the EPS programme from TTSH’s ED from 7 February 2020 (date of EPS implementation) to 20 March 2020 (date of study ethics application) inclusive. We designated a comparison cohort over a similar duration the preceding year. Relevant demographic and clinical data were extracted from the electronic medical records. Results. There was a 3.2 times higher incidence of patients with an admitting diagnosis of pneumonia from the ED in the EPS cohort compared to the comparison cohort (P<0.001). However, there was no significant difference in the median length of stay of 7 days (P=0.160). Within the EPS cohort, stroke and fluid overload occur more frequently as alternative primary diagnoses. Conclusions. Our study successfully evaluated our hospital-resource utilization demanded by our EPS programme in relation to an appropriate comparison group. This helps to inform strategic use of hospital resources to meet the needs of both COVID-19 related services and essential ‘peace-time’ healthcare services concurrently.

Additional C-type lectin receptors mediate interactions with Pneumocystis organisms and major surface glycoprotein

Introduction. Pathogen-associated molecular patterns’ (PAMPs) are microbial signatures that are recognized by host myeloid C-type lectin receptors (CLRs). These CLRs interact with micro-organisms via their carbohydrate recognition domains (CRDs) and engage signalling pathways within the cell resulting in pro-inflammatory and microbicidal responses. Gap statement. In this article, we extend our laboratory study of additional CLRs that recognize fungal ligands against Pneumocystis murina and Pneumocystis carinii and their purified major surface glycoproteins (Msgs). Aim. To study the potential of newly synthesized hFc-CLR fusions on binding to Pneumocystis and its Msg. Methods. A library of new synthesized hFc-CLR fusions was screened against Pneumocystis murina and Pneumocystis carinii organisms and their purified major surface glycoproteins (Msgs) found on the respective fungi via modified ELISA. Immunofluorescence assay (IFA) was implemented and quantified to verify results. mRNA expression analysis by quantitative PCR (q-PCR) was employed to detect respective CLRs found to bind fungal organisms in the ELISA and determine their expression levels in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model. Results. We detected a number of the CLR hFc-fusions displayed significant binding with P. murina and P. carinii organisms, and similarly to their respective Msgs. Significant organism and Msg binding was observed for CLR members C-type lectin domain family 12 member A (CLEC12A), Langerin, macrophage galactose-type lectin-1 (MGL-1), and specific intracellular adhesion molecule-3 grabbing non-integrin homologue-related 3 (SIGNR3). Immunofluorescence assay (IFA) with the respective CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of the respective CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that macrophage galactose type C-type lectin (Mgl-1), implicated in recognizing terminal N-acetylgalactosamine (GalNAc) found in the glycoproteins of microbial pathogens was significantly up-regulated during infection. Conclusion. The data herein add to the growing list of CLRs recognizing Pneumocystis and provide insights for further study of organism/host immune cell interactions.