- Volume 71, Issue 5, 2022

Tick-borne encephalitis (TBE) is caused by a neurotropic flavivirus, transmitted by the bite of Ixodes species tick vectors, and is increasing in incidence and expanding its geographical range throughout Eurasia and the Far East. Most infections are asymptomatic. However, between 2 and 30 % of cases may develop into severe neurological disease, long-term neurological sequelae or death. Diagnosis is based upon clinical signs of neurological disease and demonstration of virus-specific IgM and IgG antibodies. There is no specific antiviral treatment and supportive care is required for the various manifestations of disease. Vaccination is an effective way of preventing disease.

Louping ill virus (LIV) is a single-stranded, positive-sense RNA virus within the genus Flavivirus that is transmitted to vertebrate hosts by bites from infected ticks, the arthropod vector. The virus affects livestock in upland areas of Great Britain and Ireland, resulting in a febrile illness that can progress to fatal encephalitis. Prevention of the disease is facilitated by combining acaricide treatment, land management and vaccination strategies. However, vaccines have been discontinued in recent years. Although rare, LIV can be transmitted to and cause disease in humans. Consequently, LIV infection is a threat to human and veterinary health and can impact on the rural economy.

  Achromobacter xylosoxidans   is associated with resilient nosocomial infections, with bacteraemia, pneumonia and chronic cystic fibrosis lung infection being the most common clinical presentations. Innate multi-drug resistance and a suite of virulence factors select for   A. xylosoxidans   infection during long-term antibiotic therapy, contributing to its persistence, treatment recalcitrance, association with poor clinical outcomes and emergence as a problematic pathogen. Horizontal gene transfer and maintenance of large genomes underpin the resilience and cosmopolitan lifestyle of   A. xylosoxidans  , and complicate its phylogenetic characterization.

Loop-mediated isothermal amplification (LAMP) is a rapid alternative to PCR, in which the reaction occurs at one temperature and uses a polymerase with high displacement activity, e.g.   Bacillus stearothermophilus   DNA polymerase I (Bst) or homologues. Since the discovery of LAMP in 2000, several applications have been developed to employ this technique in the rapid detection of nucleic acid targets and enhance its performance. Improvements to the LAMP technique and a variety of innovative detection methods have led to its application for a wide range of targets in medical and veterinary microbiology, particularly in resource-poor settings. The key advantages of LAMP-based diagnostics include the ability to rapidly detect target nucleic acid sequences within 30 min and its ease of use, facilitating its application in field, bedside, pen-side, point-of-care and point-of-need diagnostic settings. LAMP can be a valuable tool to aid in the detection and management of disease outbreaks.

Introduction. Candida spp. are commensal fungal pathogens of humans, but when there is an imbalance in the microbiota, or weak host immunity, these yeasts can become pathogenic, generating high medical costs.

Gap Statement. With the increase in resistance to conventional antifungals, the development of new therapeutic strategies is necessary.

This study evaluated the in vitro antifungal activity of chlorogenic acid against fluconazole-resistant strains of Candida spp.

Mechanism of action through flow cytometry and in silico analyses, as well as molecular docking assays with ALS3 and SAP5, important proteins in the pathogenesis of Candida albicans associated with the adhesion process and biofilm formation.

Results. The chlorogenic acid showed in vitro antifungal activity against the strains tested, causing reduced cell viability, increased potential for mitochondrial depolarization and production of reactive oxygen species, DNA fragmentation and phosphatidylserine externalization, indicating an apoptotic process. Concerning the analysis through docking, the complexes formed between chlorogenic acid and the targets Thymidylate Kinase, CYP51, 1Yeast Cytochrome BC1 Complex e Exo-B-(1,3)-glucanase demonstrated more favourable binding energy. In addition, chlorogenic acid presented significant interactions with the ALS3 active site residues of C. albicans, important in the adhesion process and resistance to fluconazole. Regarding molecular docking with SAP5, no significant interactions were found between chlorogenic acid and the active site of the enzyme.

Conclusion. We concluded that chlorogenic acid has potential use as an adjuvant in antifungal therapies, due to its anti-Candida activity and ability to interact with important drug targets.

Introduction. The 16S rRNA methyltransferase (16S RMTase) gene armA is the most common mechanism conferring high-level aminoglycoside resistance in Acinetobacter baumannii , although rmtA, rmtB, rmtC, rmtD and rmtE have also been reported.

Hypothesis/Gap statement. The occurrence of 16S RMTase genes in A. baumannii in the UK and Republic of Ireland is currently unknown.

Aim. To identify the occurrence of 16S RMTase genes in A. baumannii isolates from the UK and the Republic of Ireland between 2004 and 2015.

Methodology. Five hundred and fifty pan-aminoglycoside-resistant A. baumannii isolates isolated from the UK and the Republic of Ireland between 2004 and 2015 were screened by PCR to detect known 16S RMTase genes, and then whole-genome sequencing was conducted to screen for novel 16S RMTase genes.

Results. A total of 96.5 % (531/550) of isolates were positive for 16S RMTase genes, with all but 1 harbouring armA (99.8 %, 530/531). The remaining isolates harboured rmtE3, a new rmtE variant. Most (89.2 %, 473/530) armA-positive isolates belonged to international clone II (ST2), and the rmtE3-positive isolate belonged to ST79. rmtE3 shared a similar genetic environment to rmtE2 but lacked an ISCR20 element found upstream of rmtE2.

Conclusion. This is the first report of rmtE in A. baumannii in Europe; the potential for transmission of rmtE3 to other bacterial species requires further research.

Introduction. Non-typhoidal Salmonella (NTS) bacteremia can cause significant morbidity and mortality. There is high incidence of Salmonellosis in the Middle East, including Israel, but there is a paucity of data on the clinical and epidemiological features of children in comparison to adults.

Hypothesis/Gap Statement. Previous studies describing the differences between paediatric and adult populations with Salmonella bacteremia are sparse.

Aim. This study’s aim was to describe the differences between adults and children with NTS bacteremia.

Methodology. All records of patients with NTS bacteremia between 1 January 1998 and 31 July 2020 were reviewed. Data regarding clinical manifestations and laboratory results were extracted from the medical records; records of children (aged <18 years) were compared with those of adults.

Results. Records for 137 cases of Salmonella bacteremia (69 adults and 68 children, aged 2 days to 98 years) were reviewed. Seventy nine (58 %) patients had concomitant gastrointestinal symptoms. Fifty-eight (84 %) adults and 13 (19 %) children had underlying conditions (P<0.001). Eighteen patients died, none of whom was a child. Over the study period, most of the children (n=46, 67 %) but only five adults were discharged from the emergency department on their first visit to the ED.

Conclusions. The main characteristics of NTS bacteremia in children compared to adults, are higher rates of prior discharge from emergency department, higher rate of gastrointestinal symptoms and better prognosis.

Introduction. Toxocariasis is a zoonotic parasitic disease caused by migrating nematode worms, Toxocara species larvae, within tissues. MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at a post-transcriptional level.

Hypothesis/Gap Statement. miRNA-based diagnostic biomarkers for toxocariasis are emerging, but there is limited information about the role of many miRNAs and a more detailed diagnostic evaluation of miRNA expression patterns is needed to understand their immunobiological function.

Aim. We investigated the expression levels of circulating miRNA 21 and miRNA 103a as potential biomarkers for the prediction and diagnosis of toxocariasis in Wistar rats infected with Toxocara canis.

Methodology. Thirty Wistar rats were inoculated orally with 2500 T. canis embryonated eggs via gavage. Serum samples were collected from infected animals and were tested against T. canis antigens for 60 days post-infection. The plasma samples were isolated for quantitative real-time PCR (qPCR) assays and qPCR was used to assess transcription levels of miRNA 21 and miRNA 103a.

Results. The prevalence of anti-Toxocara IgG was detected in 7/30 (23.3 %) infected rats. Molecular analysis of miRNAs 21 and 103a showed that expression levels of miRNAs in both groups of Toxocara-positive and negative samples were the same without significant association. The ratio of housekeeping gene expression (U6) to gene expression of miRNAs 21 and 103a indicated the rate of change (1/1.38 ≈ 0.75 and 1/0.751 ≈ 1.3, respectively).

Conclusion. Our study revealed that miRNAs 21 and 103a might play fundamental roles as biomarkers and diagnostic tools for toxocariasis. However, the changes in expression of these miRNAs were not adequate to be used as biomarkers in diagnosis.

Introduction. Urine samples submitted for investigation of urinary tract infection (UTI) may identify more than one bacterial isolate. These samples may be reported as ‘mixed growth urine culture’ (MGUC). The clinical significance of MGUC remains controversial.

Hypothesis/Gap Statement. The impact of MGUC on patient management is not known and should be assessed.

To describe MGUC and assess its impact on patient management.

Methodology. Microbiology laboratory reports (Leeds, UK) were retrospectively analysed and urine cultures reported as MGUC from a 1 week period underwent detailed laboratory analysis. Semi-structured interviews of NHS clinicians’ response to MGUC reports were explored for emergent themes.

Results. In 2018, 12.4 % (14,323/115,664) of urine specimens processed to detect bacterial pathogens were reported as MGUC. Among a total of 200 MGUC samples identified within 1 week in 2019, detailed laboratory analysis identified 459 bacterial isolates. Enterococcus species (30.1 %) and Escherichia coli (27.5 %) were the most frequently isolated and the most frequent organism combination (24 %). In total, 65.5 % cultures contained two organisms and 82.5 % of all MGUC contained at least one Enterobacterales . Interviews found clinicians believed MGUC reports represented detection of many commensal bacteria. Clinicians indicated they were more likely to diagnose and treat a UTI when provided with urine culture reports derived from detailed microbial analysis of MGUC, including identity and antibiotic sensitivity of organisms.

Conclusions. This study highlights the potential underuse of information derived from microbiological analysis of urine samples. Interpretive commentary on reports together with education for interpretation of enhanced reports should be explored further to improve outcomes in patients with UTI.

Introduction. Infectious keratoconjunctivitis is a significant ocular disease found in confined sheep. Little information about the aetiological agents and their antimicrobial susceptibility is available.

Gap Statement. There is limited information on the aetiological agents involved in keratoconjunctivitis outbreaks in sheep.

Aim. The present research aimed to determine the bacterial aetiological factors involved in an outbreak of infectious keratoconjunctivitis in confined lambs.

Methodology. Ocular swabs were collected from 23 randomly selected lambs, which were classified into three groups according to the severity of the lesion: group I (N=6; no ocular involvement), group II (N=8; less severe injuries) and group III (N=9; more severe injuries). Isolation of aerobic bacteria and antimicrobial susceptibility tests were carried out. Molecular detection of Mollicutes was performed, and positive samples were tested to confirm the presence of the following species: Mycoplasma conjunctivae , Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri .

Results. Moraxella sp. and Mollicutes were detected in all groups, but we inferred that Moraxella sp. are only significant in the early stages of the disease. M. conjunctivae was detected in all tested groups, while M. agalactiae was detected in samples of group III only. One strain of Moraxella sp. was resistant to erythromycin and showed intermedite resistance to tetracycline.

Conclusion. The presence of these species confirms their importance in the aetiology of this disease, and the low resistance profile observed in the studied farm suggested an increased cure success rate.

Introduction. A retrospective data analysis of 34 months (spanning 2016–2020) of 961573 diagnostic results obtained before and after nucleic acid amplification testing (NAAT) implementation, across the Public Health Wales microbiology network.

Hypothesis / Gap Statement. This is the first network-wide analysis of the implementation of enteric NAAT in diagnostic microbiology.

Aim. To assess the outcome of replacing microscopy and bacterial culture with NAAT as the primary test in the diagnosis of: Campylobacter spp., Salmonella sp., Shigella spp., Shiga toxin-producing Escherichia coli (STEC), Cryptosporidium spp. and Giardia duodenalis infections.

Methodology. Following NAAT introduction, bacterial culture was performed as a secondary test, to provide further information from NAAT positive samples for epidemiological purposes. Primary detection rates and overall bacterial culture rates were calculated for each target pathogen using both testing regimes (Stage I) including a comparison of in-patient and out-patient diagnoses (Stage II).

Results. Stage I analysis showed that the primary detection rate significantly increased for Campylobacter spp. (P<0.0001), Salmonella sp. (P=0.0151), Shigella spp. (P<0.0001), STEC (P<0.0001), Cryptosporidium spp. (P<0.0001) and Giardia duodenalis (P<0.0001) when using NAAT compared to microscopy or bacterial culture. A significant decrease was seen in the overall rate of Campylobacter spp. isolation by bacterial culture (P<0.0001), whilst other targets remained unaffected. Stage II analysis showed that NAAT positive out-patient samples were more likely to be supplemented by a positive bacterial culture than NAAT positive in-patient samples for Campylobacter spp. (P<0.0001), Salmonella sp. (P=0.0004) and STEC (P=0.0039). However, Shigella spp. was more frequently isolated from NAAT positive in-patient samples (P=0.0005). A notable increase was seen for G. duodenalis detection from in-patient samples (P=0.0002). Reference laboratory data showed the NAAT assay can detect at least 53 serotypes of STEC but may not be able to detect some of the rarer species of Cryptosporidium seen in human infections.

Conclusion. The implementation of NAAT has significantly increased the primary detection rate of all target enteric pathogens in Wales and information gleaned previously from direct culture is largely unaffected.

Introduction. Atherosclerosis is a chronic disorder in which plaque builds up in the arteries and is associated with several cardiovascular and cerebrovascular diseases such as coronary artery disease, cerebral infarction and cerebral haemorrhage. Therefore, there is an urgent need to discover new medications to treat or prevent atherosclerosis.

Hypothesis/Gap Statement. The active components of Guanxin Xiaoban capsules may have an effect on the gut microbiome of patients with atherosclerosis and have a role in their therapeutic targets.

Aim. The aim of this study was to identify genes and pathways targeted by active ingredients in Guanxin Xiaoban capsules for the treatment of atherosclerosis based on network pharmacology and analysis of changes to the gut microbiome.

Methods. Mice were treated with Guanxin Xiaoban capsules. The 16S rDNA genome sequence of all microorganisms from each group of faecal samples was used to evaluate potential structural changes in the gut microbiota after treatment with Guanxin Xiaoban capsules. Western blotting and real-time quantitative PCR were used to detect gene targets in aortic and liver tissues. Haematoxylin and eosin staining was used to observe improvements in mouse arterial plaques.

Results. The gut microbiota of atherosclerotic mice is disturbed. After Guanxin Xiaoban treatment, the abundance of bacteria in the mice improved, with an increase in the proportion of Akkermansia and a significant decrease in the proportion of Faecalibaculum . The main ingredients of Guanxin Xiaoban capsules are calycosin, liquiritin, ferulic acid, ammonium glycyrrhizate, aloe emodin, rhein and emodin. The core genes of this network were determined to be glutathione S-transferase mu 1 (GSTM1), vascular endothelial growth factor A (VEGFA) and cyclin-dependent kinase inhibitor 1A (CDKN1A). The compound–target gene network revealed an interaction between multiple components and targets and contributed to a better understanding of the potential therapeutic effects of the capsules on atherosclerosis. In addition, expression of the AGE–receptor for the AGE (RAGE) pathway was significantly inhibited and the mice showed signs of arterial plaque reduction.

Guanxin Xiaoban capsules may improve atherosclerosis and reduce the plaque area by inhibiting the AGE-RAGE signalling pathway to delay the development of atherosclerosis. This mechanism appears to involve changes in the gut microbiota. Therefore, Guanxin Xiaoban capsules have potential value as a treatment for atherosclerosis.

Introduction. Cholera is an acute enteric infection caused by Vibrio cholerae , particularly in areas lacking access to clean water. Despite the global effort to improve water quality in these regions, the burden of cholera in recent years has not yet declined. Interest has therefore extended in the use of bicistronic DNA vaccine encoding ctxB and tcpA genes of V. cholerae as a potential vaccine.

Hypothesis/Gap Statement. The potential of a bicistronic DNA vaccine, pVAX-ctxB-tcpA has not been determined in vitro and in vivo.

Aim. The goal of present study was to evaluate in vitro expression and in vivo potential of pVAX-ctxB-tcpA vaccine against V. cholerae .

Methodology. The pVAX-ctxB-tcpA was transiently transfected into mammalian COS-7 cells, and the in vitro expression was assessed using fluorescence and Western blot analyses. Next, the vaccine was encapsulated into sodium alginate using water-in-oil emulsification and evaluated for its efficiency in different pH conditions. Subsequently, oral vaccination using en(pVAX-ctxB-tcpA) was performed in vivo. The animals were challenged with V. cholerae O1 El Tor after 2 weeks of vaccination using the Removable Intestinal Tie-Adult Rabbit Diarrhoea (RITARD) model. Following the infection challenge, the rabbits were monitored for evidence of symptoms, and analysed for systemic cytokine expression level (TNF-α, IFN-γ, IL-6 and IL-10) using quantitative real-time polymerase chain reaction.

Results. The in vitro expression of pVAX-ctxB-tcpA was successfully verified via fluorescence and Western blot analyses. Meanwhile, in vivo analysis demonstrated that the en(pVAX-ctxB-tcpA) was able to protect the RITARD model against V. cholerae infection due to a lack of evidence on the clinical manifestations of cholera following bacterial challenge. Furthermore, the bicistronic group showed an upregulation of systemic IFN-γ and IL-10 following 12 days of vaccination, though not significant, suggesting the possible activation of both T-helper 1 and 2 types of response. However, upon bacterial challenge, the gene expression of all cytokines did not change.

Conclusion. Our findings suggest that the bicistronic plasmid DNA vaccine, pVAX-ctxB-tcpA, showed a potential role in inducing immune response against cholera through upregulation of in vitro gene and protein expression as well as in vivo cytokine gene expression, particularly IFN-γ and IL-10.

Introduction. Coronavirus disease 2019 (COVID-19) has caused a serious threat to public health worldwide, and there is currently no effective therapeutic strategy for treating COVID-19.

Hypothesis/Gap Statement. We propose that sophocarpine (SOP) might have potential therapeutic effects on COVID-19 through inhibiting the cytokine storm and the nuclear factor NF-κB signalling pathway.

Aim. The objective was to elucidate the potential mechanism of SOP against COVID-19 through a network pharmacology analysis and its experimental validation.

Methodology. The BATMAN-TCM database was used to identify the therapeutic targets of SOP, while the GeneCards and DisGeNET databases were used to identify the targets related to COVID-19. A protein–protein interaction (PPI) network was constructed from the STRING and analysed using Cytoscape software. Gene ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG) and disease ontology (DO) enrichment analyses of the co-targets were performed using Metascape. Autodock 4.2.6 and Pymol software were applied for molecular docking. Levels of the proinflammatory cytokines IL-6, TNFα and IL-1β were measured by ELISA, while mRNA expression levels of intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor A (VEGFA) and IFN gamma (IFNG) were detected by real-time quantitative reverse transcription PCR. The protein levels of the molecules involved in the NF-κB signalling pathway were validated by western blot analysis.

Results. A total of 65 co-targets of SOP and COVID-19 were determined. GO and KEGG enrichment analyses suggested that SOP affected COVID-19 by regulating the IL-17 signalling pathway, TNF signalling pathway and other signalling pathways. The PPI network and molecular docking showed that p65, ICAM-1 and VEGFA were key targets of SOP against COVID-19 and the underlying mechanism was validated in A549 cells in vitro. SOP attenuated the LPS-induced production of TNF-α and IL-6 and downregulated the LPS-induced mRNA expression of ICAM-1, VEGFA and IFNG. Mechanistically, SOP pretreatment inhibited the phosphorylation of p65 and facilitated the activation of Nrf2.

Conclusions. SOP has a potential therapeutic effect on COVID-19 through multiple pathways and targets, and inhibits the production of pro-inflammatory cytokines and molecules involved in the NF-κB signalling pathway.