- Volume 2, Issue 1, 2020
Volume 2, Issue 1, 2020
- Abstracts from the Microbes in Medicine Meeting 2019
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- Poster Presentation
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Salmonella antibiotic persistence and macrophage polarisation
More LessSalmonella enterica infections have a high incidence of relapse due in part to the high levels of antibiotic persistence. It is known that growing and non-growing (e.g. persister) Salmonella utilise the Salmonella Pathogenicity Island 2 (SPI-2) Type 3 Secretion System (T3SS) to dampen the pro-inflammatory M1 state of macrophages and push the immune cells towards an anti-inflammatory M2 state. This allows Salmonella prolonged survival in the host and facilitates relapse. However, this interplay between Salmonella survival and macrophage activation states remains incompletely understood.
Here, I present a study on the influence of the initial phenotypic state of untreated (M0), IFN-γ (M1) and IL-4/IL-10 (M2) prepolarised differentiated murine bone marrow-derived macrophages (MΦs) on the survival and proliferation of Salmonella enterica Serovar Typhimurium (S. Typhimurium). As expected, pro-inflammatory M1 MΦs kill S. Typhimurium more efficiently than M0 cells. Surprisingly, S. Typhimurium shows decreased survival within anti-inflammatory M2 MΦs compared to M0 MΦs, which appears to be SPI-2 dependent. Further investigation revealed that even though S. Typhimurium induces SPI-2 T3SS within M1 and M2 prepolarised MΦs, the timing of SPI-2 activation is affected during infection of M2 MΦs, thereby reducing the ability of bacteria to proliferate. This leads to many more antibiotic tolerant non-growing S. Typhimurium surviving to combined antibiotic and macrophage challenges in prepolarised MΦs.
Together, these results suggest that infection of M0 MΦs followed by reprogramming to M2 MΦs appears to promote the optimal conditions for S. Typhimurium proliferation; nevertheless, non-growing cells may immediately persist and survive better in prepolarised MΦs.
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Whole genome sequencing reveals genetic diversity in Mycobacterium avium subspecies paratuberculosis population circulating in Irish cattle
Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s Disease (JD), a chronic enteritis, in cattle. Whole genome sequencing (WGS) has been applied to many pathogen systems, where its unprecedented resolution has greatly enhanced our understanding of the molecular epidemiology of pathogen transmission. However, WGS has seen limited application to MAP; understanding the transmission dynamics of MAP will inform control of JD. We report the first study into the application of WGS to MAP in Ireland.
DNA was extracted from 167 MAP isolates sourced from cattle across Ireland. Libraries were prepared and sequenced on an Illumina-NextSeq500 platform. Sequencing data were processed using an in-house bioinformatic pipeline, which trimmed reads, aligned them to the reference genome MAP K10, followed by variant calling, quality filtering and construction of a maximum-likelihood phylogeny. DNA extracts were also used for MIRU-VNTR typing.
The resulting phylogeny shows that the MAP population present in Irish cattle is genetically diverse, which may have resulted from importation of MAP strains from across Europe into Ireland. Similar diversity was observed in a WGS study that noted the impact of cattle imports on the Canadian MAP population. Some Irish isolates were genetically similar to European and Canadian isolates.
Comparing our WGS data with MIRU-VNTR indicates that MIRU-VNTR has limited resolution for discriminating MAP strains, and often does not distinguish isolates to sufficient resolution.
The genomic data presented here provide the first snapshot of genetic diversity of Irish MAP and a baseline for future studies into spread and persistence of MAP in Irish cattle.
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Lactate improves killing of Mycobacterium tuberculosis in human macrophages
More LessMycobaterium tuberculosis (Mtb) kills more people than any other infectious agent worldwide. Our group has shown the importance of glycolysis in controlling intracellular Mtb growth in human macrophages1, which leads to increased lactate production. Therefore, we examined if lactate had an immunomodulatory effect on human monocyte derived macrophages (hMDM) infected with Mtb.
hMDM were adherence purified from healthy donor buffy coats with human serum for 6-8 days. Sodium L-Lactate was added 3 hours prior to infection with Mtb or stimulation with LPS (100 ng/ml). Macrophage phenotype and function was assessed by ELISA, flow cytometry and Seahorse metabolic-flux analysis. Bacillary killing was determined by colony forming units (CFU) after lysing infected hMDM, plating on middlebrook agar and counting 21 days later.
25 mM of lactate improves hMDM ability to control intracellular Mtb growth with a 55% reduction in CFU at day 5 post infection. The glycolytic response seen immediately following infection with Mtb or LPS stimulation was decreased with lactate. LPS-induced upregulation of activation markers was diminished by lactate.
Lactate is a product of aerobic glycolysis induced by infection and supports the intracellular killing of Mtb. Further research is required to elucidate the mechanism of this effect.
1. Gleeson, L. E. et al. Cutting Edge: Mycobacterium tuberculosis Induces Aerobic Glycolysis in Human Alveolar Macrophages That Is Required for Control of Intracellular Bacillary Replication. J. Immunol. 196, 2444–2449 (2016).
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Using metagenomics to investigate the impact of hospital stay and the ARK intervention on the human gut resistome
More LessAntimicrobials are vital for modern medicine. Antimicrobial use selects for antimicrobial resistant bacteria, particularly among the gut microflora. Minimising antimicrobial resistance (AMR) selection by avoiding unnecessary antibiotic use helps combat AMR. Metagenomic analyses have the potential to provide accurate detection and quantification of AMR genes within an individual’s gut microbiome (gut ‘resistome’), allowing the impact of different types of antibiotic exposures to be evaluated and guide interventions to reduce AMR.
We have developed a short-read sequencing approach to characterise the gut ‘resistome’ and piloted this in two clinical sample sets.
The first consisted of paired stool samples from older hospitalised adults. 25 pairs of samples (1 to 50 days apart) showed a median AMR gene reads/kb/million total reads (RPKM) of 1841 (124 to 17,832), and a median AMR gene count of 55 (2 to 101).
The second consisted of faecal discards from Clostridium difficile testing at a hospital eleven months apart. In these samples (n=21) the median AMR gene read was 923 RPKM (240 to 19,475), and the median AMR gene count was 36 (9 to 82).
In both sample sets there was a trend towards an increase in AMR gene RPKM and number between the time points. dfrF, ErmB and tetW were the commonest AMR genes in both sample sets.
This approach is being applied to analyse the impact of an intervention (ARK-Hospital) designed to change antibiotic prescribing behaviour. The data will allow us to determine the patient-level impact of reduced antibiotic exposure on carried AMR.
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The molecular basis of antibiotic treatment failure in chronic urinary tract infections
Urinary tract infections (UTIs) are amongst the most common infections worldwide, and are becoming increasingly difficult to treat. In addition to the acceleration of classic antimicrobial resistance, recurrence after initial resolution is common. Our clinical experience is that chronically infected patients sometimes fail to respond to antibiotics predicted to be effective from culture-based sensitivity testing, while antibiotics predicted to be unsuitable can succeed. We hypothesized that the bladder environment could lead to differential bacterial gene expression, resulting in differences in minimum inhibitory concentration (MICs) compared with standard culture.
Here, using strains of Escherichia coli evolved in the lab to be resistant to amoxicillin–clavulanic acid, we present data that MICs differ depending on which media the assay is performed in (M9, ISO, LB, human urine), as well as in urine-containing supernatant enriched from urothelial organoids. Next, we examined the behaviour of patient-derived Enterococcus faecalis, one of the main causative agents of chronic UTIs in the elderly. We are in the process of evaluating the MIC of first-line UTI antibiotics using growth media supplemented with urine, to more closely mimic the native uropathogen environment. Moreover, we are characterising the resistance genes expressed in those differing environments using next generation sequencing technology and comparing the results with those obtained from bacteria grown on standard diagnostic media.
Our work demonstrates the danger of extrapolating biological behaviour from artificial culture substrates and may lead to better diagnostic tests and treatments for chronic UTI.
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Socially messaging polycellular interactomes for innovative interventions in health and medicine
More LessThe fast approaching post-antibiotic era has focused efforts on the search for new classes of antimicrobial compounds to arrest the increase in mortality levels attributed to microbial infections. Key to this has been the realisation that persistent infections arise from changes in polymicrobial communities, and the interactions between the protagonists within these communities can markedly affect the effectiveness of antibiotic-based interventions. Understanding the impact of key interactions within these ecosystems is key to priming the effectiveness of drug-based interventions.
We are pursuing a chemico-biological approach to investigate the molecular mechanisms underlying the anti-infective activity of small molecules from microbe-host interactomes. Evolved through co-existence, these novel molecular entities offer an effective means of pathogen control through a form of ‘chemical messaging’. Accessing the thesaurus of microbial messaging through rational design has led to a suite of distinct bioactive frameworks that can influence inter-species behaviours, moderating chronic and acute phases of virulence and pathogenesis in Pseudomonas aeruginosa and other pathogens. Similarly, at the interkingdom level, small molecular entities with anti-inflammatory and anti-cancer activities have been identified. Elucidating the molecular mechanisms underpinning the bioactive potential of these small molecules is key to unlocking their potential to deliver next generation medicines. This can only be done with a deeper understanding of the pathoadaptive phenotypic and genotypic heterogeneity that is emerging from clinical studies.
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- Short Communication
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Antiviral activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against dengue virus type 2 (GenBank accession no. MH488959)
More LessThis study investigated the antiviral activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against dengue virus type 2 (DENV-2). The pulp of red pitahaya and the leaves of red spinach were extracted using methanol followed by sub-fractionation and Amberlite XAD16N column chromatography to obtain betacyanin fractions. The half maximum cytotoxicity concentration for betacyanin fractions from red pitahaya and red spinach on Vero cells were 4.346 and 2.287 mg ml−1, respectively. The half-maximal inhibitory concentration (IC50) of betacyanin fraction from red pitahaya was 125.8 μg ml−1 with selectivity index (SI) of 5.8. For betacyanin fraction from red spinach, the IC50 value was 14.62 µg ml−1 with SI of 28.51. Using the maximum non-toxic betacyanin concentration, direct virucidal effect against DENV-2 was obtained from betacyanin fraction from red pitahaya (IC50 of 126.70 μg ml−1; 95.0 % virus inhibition) and red spinach (IC50 value of 106.80 μg ml−1; 65.9 % of virus inhibition). Betacyanin fractions from red pitahaya and red spinach inhibited DENV-2 in vitro.
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Genotypic diversity, circulation patterns and co-detections among rhinoviruses in Queensland, 2001
More LessPurpose. Rhinoviruses (RVs) occur more frequently than other viruses and more often in people displaying symptoms than in those without. We sought to estimate the spectrum of RV diversity, RV species seasonality and to analyse RV involvement in respiratory virus co-detections.
Methodology. A convenience collection of 1179 airway sample extracts from patients with suspected respiratory infections, collected during 2001, was subjected to comprehensive molecular testing.
Results. RVs were the most common virus detected. We were able to genotype ~90 % of RV detections, identifying 70 distinct RVs, spanning all three species. RV-Bs were under-represented. We found RV species co-circulated at times, although one species usually dominated. Each species displayed a bimodal distribution.
Conclusion. Notably, RVs and influenza A viruses (IFAV) seldom co-occurred, supporting their roles as primary pathogens of the airway among acutely ill infants. Whether RV circulation has a moderating or controlling effect on the IFAV season or is controlled by it cannot be determined from these data. Despite the frequent perception that RVs commonly co-occur with another virus, our findings indicated this was not always the case. Nearly 80 % of RV detections occurred alone. Understanding more about population-level interference between viruses may allow us to harness aspects of it to generate a non-specific antiviral intervention that mimics a putative protective effect. For routine respiratory virus screening to best serve the patient, RV testing should be a principal component of any acute respiratory illness testing algorithm throughout the year.
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Prevalence and mechanisms of fluoroquinolone-resistant Escherichia coli among sheltered companion animals
More LessTo better understand the prevalence of fluoroquinolone-resistant Escherichia coli among sheltered companion animals, we conducted a screening study of 38 dogs and 78 cats and investigated the resistance mechanisms and characteristics of the isolates. Fluoroquinolone-resistant E. coli was detected in 18 dogs (47.4 %) and 14 cats (17.9 %). The isolates carried one to four mutations in the gyrA, parC and parE genes of the quinolone resistance-determining region, and the number of mutations was proportional to the MIC for ciprofloxacin. For plasmid-mediated quinolone resistance, aac-(6′)-Ib-cr was detected in nine isolates, qnrS in five isolates and qnrB in one isolate. A relationship between the presence of these genes and MIC for ciprofloxacin was not apparent. Statistical analysis indicated that fluoroquinolone-resistant E. coli was widely distributed among sheltered companion animals with various attributes. This may relate to the wide dissemination of fluoroquinolone resistance among humans and other animals in Japan.
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- Research Article
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Transcriptome analysis of Brucella abortus S19∆per immunized mouse spleen revealed activation of MHC-I and MHC-II pathways
More LessThe mouse (Mus musculus) has been extensively used for studying brucellosis, regarding pathogenesis, immunity and the evaluation of vaccines and therapeutics. In this work, RNA-seq was applied to explore the immunological potential of a live Brucella abortus S19∆per, a perosamine synthetase gene mutant of B. abortus S19. Comparison of transcriptome data was carried out for identifying differentially expressed genes among PBS (control) and B. abortus S19∆per immunized mice at 15 days post-immunization. Functional analysis revealed 545 significant differentially expressed genes related to mouse immunity. Specific activation of MHC-I and MHC-II antigen-processing pathways were identified as the highly enriched pathways based on Kyoto Encyclopedia of Genes and Genomes annotation. Other major immune response pathways regulated within the host were NF-kappa B signalling, chemokine signalling, T-cell receptor pathway, apoptosis, TNF signalling and nucleotide-binding oligomerization domain-like receptor signalling. These data provided new insights into the molecular mechanisms of B. abortus S19∆per-induced immune response in mice spleen that might facilitate the development of a highly immunogenic vaccine against brucellosis.
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Phenotypic characterization of Neisseria meningitidis strains isolated from invasive meningococcal disease in Brazil from 2002 to 2017
Introduction . Invasive meningococcal disease (IMD) has a high rate of fatality and may cause severe clinical sequelae. Over the years, the epidemiology of IMD has changed significantly in various regions of the world, and laboratory surveillance of this disease is important for mapping epidemiologic changes.
Aim. To perform phenotypic characterization of Neisseria meningitidis strains isolated from invasive disease in Brazil from 2002 to 2017, as a complementation of the data obtained in the period of 1990–2001.
Methodology . In total, 8,689 isolates sent to Adolfo Lutz Institute confirmed as N. meningitidis by conventional methods were serogrouped by slide agglutination against MenA, MenB, MenC, MenE, MenW, MenX, MenY and MenZ; serotyped and serosubtyped by a whole-cell dot-blotting assay with monoclonal antibodies.
Results . The isolates were sent from all regions of Brazil, and the southeast region was responsible for the largest number of isolates (57.2 %). Overall, the total sample (n=8,689) was represented by serogroups C (n=4,729; 54.4 %), B (n=3,313; 38.1 %), W (n=423; 4.9 %), Y (n=203; 2.3 %), X (n=5; 0.1 %) and others (n=16; 0.2 %). A shift in the prevalence of serogroups was observed in 2006, when serogroup C became the most prevalent (65.5 %), surpassing the serogroup B (21.9 %). The main isolated phenotypes were C:23:P1.14–6; B:4,7:P1.19,15; W:2a:P1.5 and W:2a:P1.5,2.
Conclusion . The data show an important change in the distribution of meningococcal serogroups, serotypes and subtypes occurring during 2002–2017. A continuous laboratory-based surveillance provides robust information to implement appropriate strategies to IMD control.
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- Case Report
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The tough process of unmasking the slow-growing mycobacterium: case report of Mycobacterium microti infection
Mycobacterium microti belongs to the Mycobacterium tuberculosis complex (MTBC). It can cause pulmonary and extrapulmonary tuberculosis in humans. Compared to M. tuberculosis , which is the most prevalent subspecies of the MTBC, M. microti infection has a different etiology. Moreover, establishing the diagnosis with conventional bacteriology can be difficult. We will illustrate this with a case of an extrapulmonary tuberculosis of the hip caused by M .microti in an immunocompetent patient in The Netherlands.
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Rare case report of infective endocarditis due to Kocuria kristinae in a patient with ventricular septal defect
More LessBackground. Infective endocarditis (IE) is an uncommon but life-threatening infection. It is commonly associated with diseased or damaged valves. Patients with congenital heart disease are more prone to getting IE than the general population. The typical organisms that cause IE include Staphylococcus , Coagulase-negative Staphylococcus, Streptococcus viridians and Enterococci. However, the importance of rare micro-organisms like Kocuria kristinae should not be underestimated especially when isolated from multiple blood cultures in patients suspected of IE.
Case presentation. We report a rare case of right-sided infective endocarditis due to K. kristinae in a young non-diabetic, non-addict female of low socioeconomic class who presented with undiagnosed fever for 1 year. She was investigated and treated for fever by several general practitioners without relief. Later on, she was diagnosed by a local cardiologist to have perimembranous ventricular septal defect with a small pulmonary valve vegetation. She was referred to a tertiary care cardiac hospital in Rawalpindi, Pakistan for further management. Transthoracic and transesophageal echocardiography confirmed IE secondary to preexisting congenital heart disease complicated with a small pulmonary vegetation. Her blood cultures yielded growth of K. kristanae, a rare micro-organism to cause IE. The patient responded to the antibiotic therapy.
Conclusion. Clinicians should have a high index of suspicion for K. kristanae IE as a possible cause of a prolonged fever especially in the presence of congenital heart disease. Antibiotic susceptibility is required for adequate therapy.
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Ignatzschineria indica bacteremia in a patient with a maggot-infested heel ulcer: a case report and literature review
More LessAn elderly patient was admitted with sepsis to a community hospital. The individual was found to have a foot wound infested with maggots, and clinical evidence of cellulitis. A blood culture was positive on day 2 with Ignatzschineria indica . The patient was treated successfully with a combination of antibiotics and manual removal of the maggots. Poor living conditions were central to his presentation.
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Prevalence and resistance pattern of uropathogens from community settings of different regions: an experience from India
Sarita Mohapatra, Rajashree Panigrahy, Vibhor Tak, Shwetha J. V., Sneha K. C., Susmita Chaudhuri, Swati Pundir, Deepak Kocher, Hitender Gautam, Seema Sood, Bimal Kumar Das, Arti Kapil, Pankaj Hari, Arvind Kumar, Rajesh Kumari, Mani Kalaivani, Ambica R., Harshal Ramesh Salve, Sumit Malhotra and Shashi Kant
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