- Volume 2, Issue 1, 2020
Volume 2, Issue 1, 2020
- Abstracts from the Microbes in Medicine Meeting 2019
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- Oral Abstract
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Experimental evolution selects clinically relevant antibiotic resistance in biofilms but with collateral tradeoffs
The widespread usage of antimicrobials in modern clinical, veterinary and industrial practices has selected for the emergence of antibiotic-resistant bacteria, which are increasingly hard to treat with currently available antibiotics. Most bacteria in nature exist in aggregated communities known as biofilms, which are inherently highly tolerant to antibiotics. There is currently a limited understanding of how biofilms evolve in response to antimicrobial pressure. Here we used a biofilm evolution model as a tool to study the effects of antimicrobial exposure on biofilms compared to planktonic cultures. We showed that biofilms of the model foodborne pathogen, Salmonella Typhimurium rapidly evolve in response to exposure to three clinically important antibiotics. Adaptation to antibiotic stress imposed a marked cost in biofilm formation, particularly evident for populations exposed to cefotaxime and azithromycin. By pairing the evolution model with whole-genome sequencing, we were able to identify and characterise two distinct mechanisms of resistance to cefotaxime and azithromycin. Among others, we identified novel substitutions within the multidrug efflux transporter, AcrB (R717L and Q176K) and validated their impact in drug export as well as changes in regulators of this efflux system. We showed that the model biofilm system selects clinically-important mechanisms of resistance and can be used to help predict how biofilms evolve under antimicrobial pressure.
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Factors governing orthologous RpoD and H-NS evolution in Salmonella enterica Serovar Typhimurium and Escherichia coli
More LessPathogenicity island acquisition is considered to be a costly process that in turn allows bacteria to explore new niches. While pathogenicity islands are known to be well integrated into existing regulatory networks in Salmonella, we do not understand whether these islands have influenced the evolution of transcription factor coding sequence itself. Here, we exchanged two antagonisticly-acting bacterial transcription factors, RpoD (σ70) and H-NS, singly or combinatorially, between the related Gram-negative bacteria Salmonella enterica Serovar Typhimurium SL1344 and Escherichia coli str. K12 substr. MG1655, to understand their functional divergence in relation to pathogenicity island expression in Salmonella. Exchanging rpoD resulted in a small growth defect in Salmonella, while exchanging hns did not. We saw a strong upregulation of pathogenicity island expression in response to rpoD exchange, while only a very weak upregulation in response to HNS exchange. Exchanging both rpoD and hns in Salmonella resulted in a further upregulation of these islands. Using two different versions of the SPI1 regulatory knockout, ΔhilA and ΔhilD, where ΔhilA knocks out only SPI1 expression and ΔhilD knocks out both SPI1 and SPI2 expression, we show that both SPI1 and SPI2 expression are costly, and that their up-regulation is the cause of the growth defect in response to rpoD exchange. Thus, we show that both RpoD and H-NS sequence evolution in Salmonella were geared towards constraining pathogenicity island expression, the expression cost of these islands very clearly being a driver of RpoD evolution.
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Short-term consumption of a high-fat diet increases host susceptibility to Listeria monocytogenes infection
A Westernized-diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice.
We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen in the gut, and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low-fat (LF) or chow-fed animals. Prior to Listeria infection short-term consumption of HF diet elevated faecal levels of Firmicutes. During active infection with L. monocytogenes microbiota changes were further exacerbated but host inflammatory responses were significantly down-regulated relative to Listeria-infected LF or chow-fed groups, suggestive of a profound tampering of the host response influenced by infection in the context of a HF diet.
Overall the results indicate that short-term consumption of a Westernized-diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease.
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Transposable temperate phages promote the evolution of divergent social strategies in Pseudomonas aeruginosa populations
More LessTransposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore over-producing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvdgenes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signaling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.
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Pharmacological modulation of the autophagy pathway enhances Mtb killing
More LessMycobacterium tuberculosis (Mtb) is the causative agent for tuberculosis which remains a serious health concern globally. The emergence of multi-drug resistant strains and the failure of BCG vaccine to control the epidemic highlight the need to achieve a better understanding of the host immune response and develop new therapies. Autophagy has been identified as an important element in both innate and adaptive immunity against tuberculosis. However Mtb employs an array of immune modulators to invade and thrive in macrophages including by inhibiting autophagosome fusion with lysosomes. Our aim is to identify novel modulators of the autophagy pathway. To do this we have screened a number of FDA approved autophagy-enhancing drugs to reverse the Mtb-induced block of autophagic flux and boost host immune responses.
Murine BMDMs expressing EGFP-LC3 were infected with H37Ra and treated with or without autophagy-enhancing drugs. The number of GFP-LC3-positive puncta per cell was quantified by high content analysis to assess autophagic induction and flux, thereby identifying compounds capable of overcoming the Mtb-induced block in autophagy. We identified three compounds, Sorafenib, Valproic Acid and Carbamazepine, which promote autophagic flux in Mtb-infected cells. Human MDMs were infected with H37Ra and treated with or without these drugs to determine their ability to promote intracellular killing of Mtb and boost the innate immune response. Our data show that Sorafenib, Valproic Acid and Carbamazepine are capable of overcoming the Mtb-induced block in autophagy and reducing bacterial burden in MDMs. This work highlights the potential for autophagy-enhancing drugs as adjunctive treatment for tuberculosis.
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The role of mature mycolic acids in mycobacterial biofilm formation and interactions with the human complement system
Tuberculosis, causative agent Mycobacterium tuberculosis, caused an estimated 10 million new cases and 1.6 million deaths in 2017.
Mycobacterial cell wall components are important for host-pathogen interactions and modulating the innate and adaptive immune response. Much is understood about the interplay between bacterial phenotype, cell wall modulation and interaction with aspects of the immune response. However, the impact of phenotype, such as biofilms on complement system activation, has not been studied in-depth.
M. tuberculosis may persist as biofilms in a chronic, non-replicating state. Mycolic acids are a major cell wall lipid and their accumulation is a hallmark of biofilm formation. M. smegmatis was mutated in gene MSMEG4722(orthologof M. tuberculosis Rv2509) (ΔMSMEG4722)which encodes a reductase involved in the final step of mycolic acid biosynthesis.
Using ΔMSMEG4722,this project explored the role of mycolic acid maturation in biofilm development, determined the relationship between lipids and complement activity with planktonic or biofilm bacteria.
Thin-layer chromatography of ΔMSMEG4722 biofilms displayed a loss of mature free mycolic acid compared to M. smegmatis, but accumulated un-reduced free mycolate. In both biofilm and planktonicΔMSMEG4722, distribution of the virulence factor trehalose dimycolate changed throughout the cell wall compared to M. smegmatis.
Flow cytometry analysis of the deposition of complement opsonin C3b revealed increased C3 on planktonic ΔMSMEG4722 compared to M. smegmatis, but this difference was not observed on bacteria from biofilms.
Evidence from this work suggests mycolic acid maturation is important for biofilm development and plays a role in complement activation on mycobacteria.
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- Poster Presentation
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Defining the link between efflux pumps and biofilm formation
More LessBiofilms are intrinsically important to our understanding of bacterial pathogens. The majority of bacterial life exists as part of a biofilm, as despite this, we have very little understanding of the genetic pathways that facilitate and drive biofilm formation. Work in our group has outlined a phenotypic link between efflux pump activity and biofilm formation, both of which have been implicated in decreased susceptibility to multiple antibiotics. Genetic or chemical inactivation of efflux results in a substantial decrease in biofilm formation. We have determined that this is due to transcriptional repression of one of the main components of the biofilm: the amyloid fibrous protein, curli. This relationship between efflux activity and biofilm formation has been identified in many Gram-negative and Gram-positive bacterial pathogens, but the regulatory network through which these phenotypes are linked is unknown. My PhD project aims to determine the pathway that links efflux pump activity and biofilm formation. I will investigate this using TraDIS, which is a large-scale transposon mutagenesis approach that will be used to determine all of the genes responsible for efflux activity and biofilm formation. My poster will present preliminary results from this genome-wide screen in E. coli. I will outline the further work to be undertaken, including repeating this experiment in Salmonella Typhimurium and formulating and testing hypotheses as to how efflux activity and biofilm formation are linked in both species. This will overall improve our understanding of important mechanisms of antimicrobial resistance in human pathogens.
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Carbapenem-resistant Enterobacteriaceae : A serious concern in cancer patients
More LessIntroduction. Gram-negative bacteria, including Enterobacteriaceae, are an important cause of infections in cancer patients. Members of Enterobacteriaceae are commonly isolated from bloodstream infections, surgical site infections, urinary tract infections, and lower respiratory tract infections. There has been an increase in the isolation of gram-negative MDRO’s over the years with ESBL’s and for the last few years with carbapenem-resistant Enterobacteriaceae (CRE). CRE’s have become a serious threat to cancer patients.
Material & Methods. A total of 61331 clinical samples from 19015 patients were received in the Dept of Microbiology, Tata Memorial Center, Mumbai, India during January 2018 to May 2019. All the samples were processed as per routine microbiological procedures and antimicrobial susceptibility testing was performed as per CLSI guidelines.
Results. Blood was the commonest sample received followed by urine, respiratory tract samples, surgical site samples and sterile body fluids. E. coli was the commonest microorganism isolated followed by Klebsiella pneumoniae, P. aeruginosa, S. aureus, Acinetobacter sp., Enterobacter sp. and Enterococci. Colistin was the most susceptible antibiotic for gram negative organisms followed by tigecycline, aminoglycosides, cefoperazone-sulbactam and piperacillin-tazobactam. Overall 77.8% CRE’s were detected whereas it was 81.8% in LRTI, followed by 82.5% in UTI, 79.1% in BSI and 75% in SSI.
Conclusion. The increasing prevalence of CRE infections represents a major threat to cancer patients. With the high mortality of CRE infections and increasing resistance to available antibiotics, it is urgent for the medical community to develop new and effective therapeutic strategies. Robust anti-microbial stewardship is the need of the hour.
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The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus
More LessRibosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase Rel Sau , with Rel Sau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway.
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The antibiofilm effects of docosahexaenoic acid
More LessThe ability of bacteria to form biofilms allows for recalcitrance against conventional antibiotic therapies (Potera, 1999). Therefore, this has contributed to the prevalence of biofilm acquired infections (BAI) clinically (Percival and Kite, 2018) which has resulted in increased morbidity and mortality amongst patients, with immunocompromised patients at risk in particular (Donlan, 2002). Biofilm formation on abiotic and biotic surfaces has enabled bacteria to colonise major organs and medical devices, such as within the lungs, on implant surfaces, contact lenses, and urinary catheters (Vinh and Embil, 2005; Percival et al., 2012; Seth et al., 2012). Docosahexaenoic acid (DHA) is a poly-unsaturated fatty acid known to exhibit antibiofilm and antimicrobial effects (Sun et al., 2017; Kim et al., 2018). Our research has found that DHA possesses strong anti-biofilm effects against Klebsiella pneumoniae and Enterococcus faecalis at low mM concentrations. DHA was capable of reducing biofilm formation by both K. pneumoniae and E. faecalis by approximately 60%. It is believed that this is the first time these effects have been reported. We have also have evidence that DHA in conjunction with an antibiotic is better at reducing biofilm formation by these strains better then either alone.
To date, there is much debate into how DHA exerts these anti-biofilm effects. DHA has been reported to distort bacterial membrane’s therefore, impacting biofilm formation (Sun et al., 2017). We aim to elucidate the precise mechanism of action by utilising a number of genetic approaches alongside the dynamic flow cell system and confocal microscopy.
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Antimicrobial resistance in bacteria associated with childhood pneumonia in Malawi
More LessCommunity acquired pneumonia (CAP) is a disease caused by an infection (by bacteria, viruses or fungi) that in cases of chronic infection lead to high mortality rates particularly in children. Pneumonia is the leading cause of death in children under the age of five worldwide with over 2 million deaths reported in 2016 by the World Health Organisation. Developing countries in sub Saharan Africa such as Malawi see some of the highest rates of the disease. CAP infections are responsible for 20% of deaths of new-borns in Malawi. Antibiotic resistance in disease causing organisms is of growing concern in countries like Malawi.
We obtained bacterial isolates from blood samples taken from Malawian children, aged 18 -60 months, diagnosed with CAP. Streptococcus pneumonia is the primary pathogen in CAP but associated ESKAPE pathogens like Pseudomonas and Staphylococci spp. and Escherichia coli are of growing importance. These were the most common bacteria purified, isolated and characterised from these samples resulting in a unique culture collection. These pathogens have been screened for antimicrobial resistance with a variety of antibiotics to generate a resistance profile based on the minimal inhibitory concentrations of selected isolates using antibiotic susceptibility disks and by broth dilution method. Initial assessment shows current front-line therapies used by the Malawian ministry of health are ineffective in treating CAP.
Our research aims to fully characterise these strains at the genetic level to better understand the mechanisms behind how antibiotic resistance develops to aid in combating this growing issue in countries like Malawi.
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Significance of the chromosomal positions of the genes encoding DNA gyrase in Salmonella enterica Serovar Typhimurium
More LessChanging positions of genes on a chromosome is an informative way of learning about why an existing chromosome structure and gene order was selected by evolution in bacteria. In Salmonella gyrA and gyrB – genes encoding DNA gyrase – enzyme that introduces negative supercoils in DNA, are located almost at opposite poles of a chromosome. However, in many other bacteria they are arranged in a gyrBA operon. In order to investigate the significance of this fact, gyrBA operon was made in Salmonella by bringing gyrA open reading frame and its ribosome binding site directly downstream of gyrB and under a control of its regulatory regions, the original copy of gyrA was deleted. The gyrBA strain obtained as a result exhibits no differences from the wild type in growth and morphology, however, the ability to supercoil DNA is altered between gyrBA and the WT. This is specifically important at conditions mimicking environment inside a macrophage in terms of Mg2+ concentration, as it may suggest alteration of gyrBA survival inside a macrophage. An attempt to make strain with gyrAB operon was not successful, as it was not possible to delete the original gyrB, suggesting particular importance of its position.
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Understanding selection of antimicrobial resistance in biofilms through experimental evolution
More LessBackground. Biofilms possess several important properties which differentiate them from free-living cells, including their response to selective pressures. This work demonstrates a link between exposure to non-therapeutic antimicrobials and selection for antibiotic resistance in experimentally evolved biofilms of Pseudomonas aeruginosa.
Methods. Biofilms of P. aeruginosa PA14 were experimentally evolved through successive subculture under periodically increasing antimicrobial stress. Beginning at 0.25-times MIC, biofilms were exposed to benzalkonium chloride, ciprofloxacin, colistin, zinc or copper sulfate. When the antimicrobial stress resulted in growth failure, the last successful passage was phenotyped. Biofilm formation was quantified via crystal violet uptake and MICs determined by broth microdilution according to EUCAST guidelines.
Results. Experimental evolution selected for mutants which produced approximately four-fold more biomass than the parenteral strain. Both ciprofloxacin and chloramphenicol readily selected for mutants able to survive >1024-times MIC. Conversely, no change in susceptibility was observed for either metal. However, despite zinc sulfate not selecting for decreased susceptibility to itself, cross-resistance to both colistin and meropenem was observed in the zinc-passaged lineages. Benzalkonium chloride did not select for a significant change in susceptibility when grown planktonically. However, when assayed as a biofilm, a 5-fold decrease in benzalkonium chloride susceptibility was observed, indicating a biofilm-specific mechanism of antimicrobial tolerance.
Conclusion. Mutants with an increased capacity to form biofilms and decreased antimicrobial susceptibility were successfully selected. Furthermore, zinc sulfate, a common feed additive, was able to select for decreased susceptibility to clinically-relevant antibiotics, lending to concerns that ubiquitous non-therapeutic antimicrobials may act as non-canonical drivers of antibiotic resistance.
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The evolution of Pseudomonas aeruginosa during short-term acute respiratory infections
Background. The emergence and spread of bacteria with resistance to multiple antimicrobial agents is raising the mortality rates associated with bacterial infections, and poses a fundamental threat to human health. Pseudomonas aeruginosais an opportunistic human pathogen that is responsible for 10-15% of health-care associated infections worldwide, and is known for its ability to rapidly develop enhanced resistance during the course of treating an infection.
Methods. P. aeruginosa isolates have been collected from ICU patients in hospitals across Europe over a longitudinal sampling of short-term acute respiratory infection. Twelve isolates per patient per sampling of infection have been acquired. Genome sequencing and high-throughput phenotyping of growth rate and antibiotic resistance profile has been carried out.
Results and conclusions. Using high-throughput phenotyping and genome sequence data we have been able to characterise population diversity and variation in phenotypic resistance profile, growth rate and acquired resistance gene content of these isolates. A gradient of diversity was found within patient infections, ranging from monoclonal to multiple strains present. Large within-patient population diversity in phenotypic resistance profile and acquired resistance gene content could be observed in multiple sequence-type infections.
This research has been carried out in collaboration with the COMBACTE-MAGNET research consortium (Combatting Bacterial Resistance in Europe – Molecules against Gram-Negative Infections).
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Plasma activated liquids: New decontamination solutions
More LessIn the field of new decontaminants, there is an increasing consensus that improved disinfection of environmental surfaces is needed in patient care facilities as an important component in the overall strategy for prevention of HAI. The interaction of cold atmospheric plasma, i.e. an ionized gas, with liquids results in complex physical and chemical processes, which offer a source of short-lived and long-lived reactive chemical species that are critical for microbial inactivation. These solutions may fulfil the urgent need for new decontaminant solutions for special purposes such as disinfection of surfaces, or use as an antiseptic for body surfaces.
In this study, we explored the bactericidal effects of plasma activated liquids (PALs) on E.coli and S. aureus strains and investigated factors which influence PALs stability over time. Liquids of interest were non-complex solutions such as water and saline. An atmospheric cold plasma system using air was employed for the generation of PALs. The solutions were compared with respect to their content of long-lived reactive chemical species and bactericidal effects.
Our results documented that PALs may carry different concentrations of chemical species and maintain diverse antimicrobial properties. The bactericidal activity of these solutions demonstrated high thermal stability and could be preserved over a 6 month period through specific sub-ambient storage conditions. Investigation of the inactivation processes in relation to the PALs’ chemical composition will enhance our knowledge on how prokaryotic and eukaryotic cells respond to these, and demonstrate how PALs could be a promising treatment method for future applications, with chemical and antimicrobial stability.
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A matter of life and death; identifying host genomic factors that determine susceptibility of chickens to highly pathogenic avian influenza
More LessDNA samples collected from survivors of recent outbreaks of highly pathogenic avian influenza (HPAI) in Mexico and the USA have provided a rare opportunity to study the genetic mechanisms underpinning susceptibility of chickens to this devastating and economically impactful disease which normally exhibits 70-100% mortality in the chicken host. Whole Genome Sequence (WGS) data has been used to perform Genome-Wide Association Studies (GWAS), which have highlighted single nucleotide polymorphisms (SNPs) segregating significantly between survivors and controls, with a pedigreed experimental group exhibiting a highly significant signal on chicken chromosome 2 in the region of a biologically relevant gene. Candidate SNPs for resilience are currently being validated using in vitro gene editing methodologiesthat modulate candidate gene expression to investigate the effect on viral replication and cellular response to HPAI infection. A detailed understanding of the genomic resilience to HPAI from this study will have implications for both the poultry industry and for public health.
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Characterising the urinary microbial community in patients with symptoms of urinary tract infection
More LessBackground. Every year 250 million individuals develop urinary tract infections (UTI), the diagnosis of which, usually involves the ‘gold standard’ midstream urine culture (MSU) test. Prompt detection aided by sensitive tests may increase the likelihood of successful treatment. However, recent evidence highlights the potential inadequacies of this test.
Methods. We assessed the UK’s MSU culture by comparison with enhanced culture techniques and 16S rRNA gene sequencing. With ethical approval, patients attending their first consultation at the Whittington Hospital Clinic and asymptomatic controls provided MSU specimens. Each specimen was submitted to the Hospital Microbiology laboratory for MSU culture. 16S rRNA gene sequencing was performed on 1ml unspun urine and 30ml of centrifuged urine.
Results. Urine specimens were analysed for 33 patients (mean age= 49 years, sd=16.5) and 29 controls (mean age=40.7 years, sd=15.7). Comparison of routine MSU cultures between patients and controls indicated that the test failed to discriminate between patients and controls(χ2= 0.539, df = 1, P = 0.674). Bacterial DNA was detected in 97.0% patients and 89.7% controls. Enterobacteriaceae was most abundant in patients, whereas Streptococcus dominated in controls. A higher distribution of the median number of observed taxa (centrifuged and unspun samples combined) was seen in patients (χ2 = 8.0, df = 2, P <0.05).
Conclusion. The MSU culture failed to discriminate between patients and controls and missed recognised uropathogens that were detected with bacterial DNA sequencing. Microbial communities characterised by sequencing warrant further investigation to understand the role of each member in UTI.
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Vorinostat (SAHA) promotes innate and adaptive immunity to Mycobacterium tuberculosis
More LessMycobacterium tuberculosis (Mtb) is increasingly resistant to antibiotics. Therefore, it is paramount to develop host-directed therapies (HDT) aimed at activating the host immune response to promote bacterial killing. Mtb is phagocytosed by alveolar macrophages (AM) and infiltrating monocyte-derived macrophages (MDM) in the lung which upregulate effector functions by epigenetic modifications to enable transcription. Suberanilohydroxamic acid (Vorinostat; SAHA), an FDA-approved histone deacetylase inhibitor, can modulate these changes and support immunity.
Human AM were purified from bronchoalveolar lavage. MDM were obtained from blood of healthy donors and patients with TB. Macrophages were infected with Mtb in the presence of SAHA. After 24 hours, cytokine secretion was quantified. Macrophages were washed and co-cultured with CFSE-labelled PBMC from IGRA positive donors. T-cell responses were analysed by flow cytometry and ELISA. Macrophages were lysed and colony forming units were enumerated.
SAHA increased IL-1β and decreased IL-10 in human AM and MDM infected with Mtb. Proliferating T-helper cells co-cultured with Mtb-infected, SAHA-treated AM or MDM exhibited enhanced IFN-γ and GMCSF co-production. SAHA promotes proinflammatory immune responses to Mtb infection in human AM and MDM, with a subsequent effect on T cell responses. SAHA may therefore be beneficial as a host-directed therapy or vaccine adjunct against TB.
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Development of a microbially-derived therapy against Fusobacterium nucleatum, a bacterial pathogen linked with colorectal cancer
More LessBackground. Specific bacterial species have been linked to several intestinal diseases, including colorectal cancer (CRC). In recent years, high abundances of an emerging pathogen, Fusobacterium nucleatum, have been identified in tumors and stool samples of CRC patients and it has been suggested that F. nucleatum contributes to CRC initiation and development. The possibility of suppressing the growth of F. nucleatum in the GI tract using antimicrobial-producing probiotic bacteria may reduce the overall risk of CRC development.
Methods. Here, we screen a collection of faecal samples from healthy donors against F. nucleatum in an effort to discover an antimicrobial-producing isolate capable of selectively inhibiting this emerging human pathogen. Potential isolates with anti-Fusobacterial activity were then further analysed for the ability to inhibit the pathogen in cell culture and in a faecal fermentation system, which simulates the dynamic conditions of the human colon.
Results. Culture-based screening of over 16,000 colonies of gastrointestinal origin resulted in the identification of one faecal isolate with probiotic potential displaying significant antagonistic activity against F. nucleatum initially in cell culture media and subsequently inhibition was confirmed in the simulated intestinal model.
Conclusion. This study reveals that, a novel gut isolate demonstrates inhibition against the CRC-associated F. nucleatum in vitro and suppresses its growth in a model of the human distal colon. This is an important finding, suggesting the potential of a natural gut bacterium to supress the growth of a bacterial pathogen associated with CRC, which may contribute to reducing the overall risk of developing the disease.
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Quorum quenching activity of Aerva lanata against Catheter Associated Urinary Tract Infections (CAUTI) Pathogens
More LessBackground. CAUTI is the most common hospital infection caused by Gram-negative bacterium owing to their ability to colonize UT mucosa. These uro-pathogens elaborate quorum sensing (QS) molecules that modify the expression and secretion of biofilm matrix compounds in response to rheological and other UT changes during catheterization. Quenching the biological effects of QS molecules will have substantial clinical value. This study identifies and evaluate the antibiofilm/anti-virulence properties of Aerva lanata plant extract against select CAUTI pathogens.
Methods. Methanolic extract of Aerva lanata was screened for its anti-urobacterial effects using Chromobacterium violaceum as the test strain. A. lanata extract impact on the QS molecule, acyl homoserine lactone (AHL) regulated bacterial functions essential for biofilm formation was investigated. It includes RT-PCR analysis of QS genes in the bacterium and in silico studies of bacterial compounds.
Results. Methanolic extract of A. lanata interfered with AHL regulated physiological functions such as biofilm formation, pellicle inhibition, flagellar motility and exopolysaccharides (EPS) production. In situ-visualization of biofilm with confocal microscopy showed concentration influenced reduction in bacterial biofilm formation in response to plant extract. GC-MS identified 11 compounds and molecular docking analysis predicted their respective host receptors. In silico analysis of plant extract revealed putative compounds with potential to inhibit the bacterial QS AHL system. RT-PCR analysis revealed down regulation of QS related virulence genes viz., las1, lasR, lasB, lasA.
Conclusion. Data obtained in this study strongly suggests that Aerva lanata should be further investigated for its therapeutic potentials to treat bacterial CAUTI.
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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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